Serpina1 sirnas: compositions of matter and methods of treatment

ABSTRACT

The technology described herein relates to double-stranded ribonucleic acid (dsRNA) compositions targeting the Serpina1 gene, and methods of using such dsRNA compositions to inhibit expression of Serpina1.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of co-pending U.S. application Ser.No. 14/974,650 filed Dec. 18, 2015 which is a continuation of Ser. No.14/127,782, filed Dec. 19, 2013 now abandoned, which is a 35 U.S.C. §371 National Phase Entry Application of International Application No.PCT/US2012/043782 filed Jun. 22, 2012, which designates the U.S., andwhich claims benefit under 35 U.S.C. § 119(e) of U.S. ProvisionalApplication Nos. 61/500,387 filed Jun. 23, 2011, 61/509,974 filed Jul.20, 2011 and 61/608,698 filed Mar. 9, 2012, the contents of which areincorporated herein by reference in their entireties.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted in ASCII format via EFS-Web and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Dec. 19, 2013, isnamed 05105807.txt and is 146,610 bytes in size.

TECHNICAL FIELD

The technology described herein relates to the specific inhibition ofgene expression.

BACKGROUND

Serpina1 (α-1 antitrypsin or AAT) is an inhibitor of neutrophil elastaseproduced by hepatocytes, mononuclear monocytes, alveolar macrophages,enterocytes, and myeloid cells. Individuals with mutations in one orboth copies of the Serpina1 gene can suffer from alpha-1 anti-trypsindeficiency, which presents as a risk of developing pulmonary emphysemaor chronic liver disease due to greater than normal elastase activity inthe lungs and liver.

In affected individuals, the deficiency in alpha-1 antitrypsin is adeficiency of wildtype, functional alpha-1 antitrypsin. In some cases,the individual is producing significant quantities of alpha-1antitrypsin, but a proportion of the alpha-1 antitrypsin protein beingproduced is misfolded or contains mutations that compromise thefunctioning of the protein. In some cases, the individual is producingmisfolded proteins which cannot be properly transported from the site ofsynthesis to the site of action within the body. Liver disease resultingfrom alpha-1 antitrypsin deficiency can be caused by such misfoldedproteins. Mutant forms of alpha-1 antitypsin are produced in liver cellsand in the misfolded configuration they are not readily transported outof the cells. This leads to a buildup of misfolded protein in the livercells and can cause one or more diseases or disorders of the liverincluding, but not limited to, chronic liver disease, liverinflammation, cirrhosis, liver fibrosis, and/or hepatocellularcarcinoma.

There are currently very limited options for the treatment of patientswith liver disease arising from alpha-1 antitrypsin deficiency,including hepatitis vaccination, supportive care, avoidance of injuriousagents (e.g. alcohol and NSAIDs). Replacement of alpha-1 antitrypsin hasno impact liver disease in these patients but liver transplantation canbe effective. Provided herein are methods for treating or preventingchronic liver disease due to Serpina1 deficiency using inhibitory RNAs(iRNAs).

Double-stranded RNA molecules (dsRNA) have been shown to block geneexpression in a highly conserved regulatory mechanism known as RNAinterference (RNAi). WO 99/32619 (Fire et al.) disclosed the use of adsRNA of at least 25 nucleotides in length to inhibit the expression ofgenes in C. elegans. dsRNA has also been shown to degrade target RNA inother organisms, including plants (see, e.g., WO 99/53050, Waterhouse etal.; and WO 99/61631, Heifetz et al.), Drosophila (see, e.g., Yang, D.,et al., Curr. Biol. (2000) 10:1191-1200), and mammals (see WO 00/44895,Limmer; and DE 101 00 586.5, Kreutzer et al.). This natural mechanismhas now become the focus of a new class of pharmaceutical agents fortreating disorders that are caused by the aberrant or unwantedregulation of a gene.

SUMMARY

Described herein are compositions and methods that effect theRNA-induced silencing complex (RISC)-mediated cleavage of RNAtranscripts of the Serpina1 gene, such as in a cell or mammal. Alsodescribed are compositions and methods for treating alpha-1 anti-trypsinrelated liver disease and related pathological conditions caused by theexpression of a Serpina1 gene, (e.g. chronic liver disease,inflammation, fibrosis and/or cirrhosis).

As used herein, the term “iRNA” refers to an agent that contains RNA asthat term is defined herein, and which mediates the targeted cleavage ofan RNA transcript via an RNA-induced silencing complex (RISC) pathway.In one embodiment, the iRNA inhibits the expression of Serpina1 in acell or mammal. Alternatively, in another embodiment, the iRNAup-regulates the expression of Serpina1 in a cell or mammal.

The iRNAs included in the compositions featured herein encompass a dsRNAhaving an RNA strand (the antisense strand) having a region that is 30nucleotides or less, generally 19-24 nucleotides in length, that issubstantially complementary to at least part of an mRNA transcript of aSerpina1 gene. In one embodiment, the dsRNA comprises a region of atleast 15 contiguous nucleotides.

In one embodiment, an iRNA for inhibiting expression of a Serpina1 geneincludes at least two sequences that are complementary to each other.The iRNA includes a sense strand having a first sequence and anantisense strand having a second sequence. The antisense strand includesa nucleotide sequence that is substantially complementary to at leastpart of an mRNA encoding Serpina1, and the region of complementarity is30 nucleotides or less, and at least 15 nucleotides in length.Generally, the iRNA is 19 to 24, e.g., 19 to 21 nucleotides in length.In some embodiments the iRNA is from about 15 to about 25 nucleotides inlength, and in other embodiments the iRNA is from about 25 to about 30nucleotides in length. The iRNA, upon contacting with a cell expressingSerpina1, inhibits the expression of a Serpina1 gene by at least 10%, atleast 20%, at least 25%, at least 30%, at least 35% or at least 40% ormore, such as when assayed by a method as described herein. In oneembodiment, the Serpina1 iRNA is formulated in a stable nucleic acidlipid particle (SNALP).

In one embodiment, an iRNA featured herein includes a first sequence ofa dsRNA that is selected from the group consisting of the sensesequences of Tables 3 and/or 4, and a second sequence that is selectedfrom the group consisting of the corresponding antisense sequences ofTables 3 and/or 4. The iRNA molecules featured herein can includenaturally occurring nucleotides or can include at least one modifiednucleotide, including, but not limited to a 2′-O-methyl modifiednucleotide, a nucleotide having a 5′-phosphorothioate group, and aterminal nucleotide linked to a cholesteryl derivative. Alternatively,the modified nucleotide can be chosen from the group of: a2′-deoxy-2′-fluoro modified nucleotide, a 2′-deoxy-modified nucleotide,a locked nucleotide, an abasic nucleotide, 2′-amino-modified nucleotide,2′-alkyl-modified nucleotide, morpholino nucleotide, a phosphoramidate,and a non-natural base comprising nucleotide. Generally, such a modifiedsequence will be based on a first sequence of said iRNA selected fromthe group consisting of the sense sequences of Tables 3 and 4 and asecond sequence selected from the group consisting of the antisensesequences of Tables 3 and 4.

In some embodiments, an iRNA featured herein can comprise 2′-O-methylpyrimidines (e.g. 2′ 0-Methyl C and 2′-O-Methyl U). In some embodiments,an iRNA featured herein comprises 2′-O-methyl pyrimidine modification ofevery pyrimidine comprised by the iRNA. In some embodiments, an iRNAfeatured herein can comprise 2′-O-methyl pyrimidine modification ofevery pyrimidine comprised by one strand (e.g. the sense or theantisense strand) of the iRNA. In some embodiments, an iRNA featuredherein can comprise 2′-O-methyl pyrimidine modification of a pyrimidineadjacent to a ribo A nucleoside. In some embodiments, an iRNA featuredherein can comprise 2′-O-methyl pyrimidine modification of pyrimidinesadjacent to a ribo A nucleoside on one strand of the iRNA. In someembodiments, an iRNA featured herein can comprise 2′-O-methyl pyrimidinemodification of a pyrimidine immediately 5′ of a ribo A nucleoside. Insome embodiments, an iRNA featured herein can comprise 2′-O-methylpyrimidine modification of pyrimidines immediately 5′ of a ribo Anucleoside by the iRNA on one strand of the iRNA. In some embodiments,an iRNA featured herein can comprise a two base dTsdT extension at the3′ end of at least one strand of the iRNA. In some embodiments, an iRNAfeatured herein can comprise a two base dTsdT extension at the 3′ end ofboth strands of the iRNA.

In one embodiment, the subject is selected, at least in part, on thebasis of needing a reduction in misfolded Serpina1 protein, a reductionin misfolded Serpina1 protein in the liver, or an increase in wild-typeplasma Serpina1 protein. In certain embodiments, the patient in need ofa treatment for a disorder mediated by Serpina1 expression has thesymptoms of, is diagnosed with, and/or is at risk for developing alpha-1anti-trypsin related liver disease, chronic liver disease, liverinflammation, cirrhosis, liver fibrosis, and/or hepatocellularcarcinoma.

In one embodiment, an iRNA as described herein targets a wildtypeSerpina1 RNA transcript, and in another embodiment, the iRNA targets amutant transcript (e.g., a Serpina1RNA carrying an allelic variant). Forexample, an iRNA of the technology described herein can target apolymorphic variant, such as a single nucleotide polymorphism (SNP)variant, of Serpina1 (e.g. the PiZ SNP (NCBI ID NO: dbSNP:rs28929474),the PiW SNP (NCBI ID NO: dbSNP:rs1802959, the PiNull(Devon) SNP (NCBI IDNO: dbSNP:rs11558261), and/or the PiNull(Ludwigshafen) SNP (NCBI ID NO:dbSNP:rs28931572). In a further example, an iRNA of the technologydescribed herein can target a polymorphic variant, such as a mutantallele of Serpina1 that encodes a mutant mRNA (e.g. the PiM(Malton)allele (see Frazier et al., Am J Hum Genet. 1989 44:894-902; Curiel etal., J Biol Chem 1989 264:13938-45; Graham et al., Hum Genet. 198984:55-8). In another embodiment, the iRNA targets both a wildtype and amutant Serpina1 transcript. In yet another embodiment, the iRNA targetsa transcript variant of Serpina1.

In one embodiment, an iRNA featured in the technology described hereintargets a non-coding region of a Serpina1 RNA transcript, such as the 5′or 3′ untranslated region.

In one aspect, the technology described herein provides a cellcontaining at least one of the iRNAs featured in the technologydescribed herein. The cell is generally a mammalian cell, such as ahuman cell.

In another aspect, the technology described herein provides apharmaceutical composition for inhibiting the expression of a Serpina1gene in an organism, generally a human subject. The compositiontypically includes one or more of the iRNAs described herein and apharmaceutically acceptable carrier or delivery vehicle. In oneembodiment, the composition is used for treating a disorder mediated bySerpina1 expression. In another embodiment, the composition is used fortreating alpha-1 anti-trypsin related liver disease. Ian anotherembodiment, the composition is used to reduce the severity, signs,symptoms, and/or markers of cirrhosis, fibrosis, Serpina1 accumulationin the liver and/or liver inflammation. In another embodiment, thecomposition is used to decrease the risk of developing cirrhosis,fibrosis, Serpina1 accumulation in the liver, hepatocellular carcinoma,and/or liver inflammation.

In another embodiment, the pharmaceutical composition is formulated foradministration of a dosage regimen described herein, e.g., not more thanonce every four weeks, not more than once every three weeks, not morethan once every two weeks, or not more than once every week. In anotherembodiment, the administration of the pharmaceutical composition can bemaintained for a month or longer, e.g., one, two, three, or six months,or one year or longer.

In another embodiment, a composition containing an iRNA featured in thetechnology described herein, e.g., a dsRNA targeting Serpina1, isadministered with a non-iRNA therapeutic agent, such as an agent knownto treat a liver disorder, or a symptom of a liver disorder. Forexample, an iRNA featured in the technology described herein can beadministered with an agent for treatment of cirrhosis or other disordersassociated with alpha-1 anti-trypsin related liver disease.

In another embodiment, a Serpina1 iRNA is administered to a patient, andthen the non-iRNA agent is administered to the patient (or vice versa).In another embodiment, a Serpina1 iRNA and the non-iRNA therapeuticagent are administered at the same time.

In another aspect, the technology described herein provides a method forinhibiting the expression of a Serpina1 gene in a cell by performing thefollowing steps:

-   -   (a) introducing into the cell a double-stranded ribonucleic acid        (dsRNA), wherein the dsRNA includes at least two sequences that        are complementary to each other. The dsRNA has a sense strand        having a first sequence and an antisense strand having a second        sequence; the antisense strand has a region of complementarity        that is substantially complementary to at least a part of an        mRNA encoding Serpina1, and where the region of complementarity        is 30 nucleotides or less, i.e., 15-30 nucleotides in length,        and generally 19-24 nucleotides in length, and where the dsRNA,        upon contact with a cell expressing Serpina1, inhibits        expression of a Serpina1 gene by at least 10%, preferably at        least 20%, at least 30%, at least 40% or more; and    -   (b) maintaining the cell produced in step (a) for a time        sufficient to obtain degradation of the mRNA transcript of the        Serpina1 gene, thereby inhibiting expression of a Serpina1 gene        in the cell.

In another aspect, the technology described herein provides methods andcompositions useful for activating expression of a Serpina1 gene in acell or mammal.

In another aspect, the technology described herein provides a method formodulating the expression of a Serpina1 gene in a cell by performing thefollowing steps:

-   -   (a) introducing into the cell a double-stranded ribonucleic acid        (dsRNA), wherein the dsRNA includes at least two sequences that        are complementary to each other. The dsRNA has a sense strand        having a first sequence and an antisense strand having a second        sequence; the antisense strand has a region of complementarity        that is substantially complementary to at least a part of an        mRNA encoding Serpina1, and where the region of complementarity        is 30 nucleotides or less, i.e., 15-30 nucleotides in length,        and generally 19-24 nucleotides in length, and where the dsRNA,        upon contact with a cell expressing Serpina1, inhibits        expression of a Serpina1 gene by at least 10%, preferably at        least 20%, at least 30%, at least 40% or more; and    -   (b) maintaining the cell produced in step (a) for a time        sufficient to obtain degradation or protection of the mRNA        transcript of the Serpina1 gene, thereby modulating expression        of a Serpina1 gene in the cell.

In one embodiment, the method is for inhibiting gene expression in aliver cell, a monocyte, an alveolar macrophage, an enterocyte, and/or amyeloid cell. In another embodiment, the method is for activating geneexpression in a liver cell, a monocyte, an alveolar macrophage, anenterocyte, and/or a myeloid cell.

In other aspects, the technology described herein provides methods fortreating, preventing or managing pathological processes mediated bySerpina1 expression, such as an alpha-1 anti-trypsin deficiency liverdisease. In one embodiment, the method includes administering to apatient in need of such treatment, prevention or management atherapeutically or prophylactically effective amount of one or more ofthe iRNAs featured in the technology described herein. In one embodimentthe patient has chronic liver disease. In another embodiment,administration of the iRNA targeting Serpina1 alleviates or relieves theseverity of at least one symptom of a Serpina1-mediated disorder in thepatient, such as liver inflammation.

In one aspect, the technology described herein provides a vector forinhibiting the expression of a Serpina1 gene in a cell. In oneembodiment, the vector includes at least one regulatory sequenceoperably linked to a nucleotide sequence that encodes at least onestrand of an iRNA as described herein.

In another aspect, the technology described herein provides a cellcontaining a vector for inhibiting the expression of a Serpina1 gene ina cell. The vector includes a regulatory sequence operably linked to anucleotide sequence that encodes at least one strand of one of the iRNAsas described herein.

In yet another aspect, the technology described herein provides acomposition containing a Serpina1 iRNA, in combination with a secondiRNA targeting a second gene involved in a pathological disease, anduseful for treating the disease, e.g., a liver disorder.

The details of various embodiments of the invention are set forth in thedescription below. Other features, objects, and advantages of theinvention will be apparent from the description and the drawings, andfrom the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph showing the inhibition of Serpina1 expression in Hep3Bcells following a single dose of the indicated iRNAs. Duplex IDs areshown on the x-axis and expression normalized to cells transfected withmock-inoculated and cells inoculated with the AD-1955 (10 nM) control isshown on the y-axis. Duplex IDs in the shaded boxes were selected forIC₅₀ tests.

FIG. 2 is a graph comparing inhibition of expression of wild-typeSerpina1 and the PiZ allele in 293T cells. Duplex AD-44715 was used atthe concentrations shown on the x-axis. Serpina1 expression is show inarbitrary units after normalizing to GAPDH expression

FIG. 3 is a graph of in vivo tests of Serpina1 expression inhibitionusing Duplex AD-44715. After a first injection of plasmids expressinghuman Serpina1 (wildtype (WT_mycAAT) or PiZ (Z-mycAAT)) or a control,AD-44715 and AD-1955 control iRNAs were delivered to mice intravenouslyand expression of Serpina1 detected after 48 hours. Treatments areindicated on the x-axis while the y-axis shows Serpina1 expression inarbitrary units following normalization to GAPDH.

FIG. 4 depicts a graph of the normalized serum level of Z-AAT intransgenic animals 48 hours after intravenous administration of iRNAs.PBS=phosphate buffered saline control; LUC=AD-1955 iRNA control,AAT=AD-44715 iRNA. Doses as shown. The y-axes show the level of humanAAT mRNA normalized to mouse GAPDH mRNA. The value of each bar is theaverage level for that experimental group. The value of each bar isshown above the bar.

FIG. 5 depict a graph of the normalized serum level of Z-AAT intransgenic animals 48 hours after intravenous administration of iRNAs.PBS=phosphate buffered saline control; LUC=AD-1955 iRNA control,AAT=AD-44715 iRNA. Doses as shown. The y-axes show the level of humanAAT protein in mg/L.

FIGS. 6A-6B depict a Western blot and graph representing quantitation ofthe Western blot of the level of AAT monomer in the livers of transgenicanimals 48 hours after intravenous administration of iRNAs.PBS=phosphate buffered saline control; LUC=AD-1955 iRNA control,AAT=AD-44715 iRNA; mpk=mg/kg; RDU=relative denisometric units. FIG. 6Ais a Western blot detecting AAT monomer levels. FIG. 6B is a graph ofthe level of AAT monomer for each experimental group.

FIGS. 7A-7B depict a Western blot and graph representing quantitation ofthe Western blot of the level of AAT polymer in the livers of transgenicanimals 48 hours after intravenous administration of iRNAs.PBS=phosphate buffered saline control; LUC=AD-1955 iRNA control,AAT=AD-44715 iRNA; mpk=mg/kg; RDU=relative denisometric units. FIG. 7Ais a Western blot detecting AAT polymer levels. FIG. 7B is a graph ofthe level of AAT polymer for each experimental group. The values shownabove the bars in 7B are the relative densities of each spot wherein theLuc control is set to an arbitrary value of 100.

FIGS. 8A-8C depict Western blots and graphs representing quantitation ofthe Western blots of the level of AAT in the livers of transgenicanimals 48 hours after intravenous administration of iRNAs.PBS=phosphate buffered saline control; LUC=AD-1955 iRNA control,AAT=AD-44715 iRNA; mpk=mg/kg; RDU=relative denisometric units. FIG. 8Ais a Western blot detecting AAT monomer levels. FIG. 8B is a Westernblot detecting AAT polymer levels. FIG. 8C is a graph of the ratio ofAAT monomer:AAT polymer for each experimental group.

FIGS. 9A-9B depict graphs representing quantitation by RT-PCR andWestern blots measuring the level of AAT in the serum and livers oftransgenic animals 48 hours after intravenous administration of iRNAs.PBS=phosphate buffered saline control; LUC=AD-1955 iRNA control,AAT_2=AD-44724 iRNA; mpk=mg/kg; RDU=relative densitometric units. FIG.9A is a graph of the level of AAT mRNA in the livers of the animals.FIG. 9B is a graph of the level of AAT protein in the serum of theanimals.

FIGS. 10A-10C depict a diagram and graphs of a multiple-dose iRNAexperiment. FIG. 10A is a diagram of the experimental procedure.Sac=sacrifice. FIG. 10B is a graph representing AAT mRNA levels in thelivers of transgenic animals after intravenous administration of iRNAs.FIG. 10C is a graph representing quantitation of Western blots measuringthe level of AAT in the serum of transgenic animals after intravenousadministration of iRNAs. PBS=phosphate buffered saline control; Controlor siLUC=AD-1955 iRNA control, siAAT or “drug”=AD-44715 iRNA; mpk=mg/kg;RDU=relative densiometric units.

FIGS. 11A-11E depict a diagram and graphs of a multiple-dose AAT iRNAdosing experiment and graphs and Western blots showing the resultsthereof. FIG. 11A is a diagram of the experimental procedure.Sac=sacrifice. FIG. 11B is a graph representing quantitation of Westernblots measuring the level of AAT momomeric form in the livers oftransgenic animals administered control and AAT-specific siRNAformulations after sacrifice under the experimental protocol set out inFIG. 11A. Units on the y-axis are RDU (relative densiometric units).FIG. 11C shows a Western blot for the monomeric form of AAT in the liverat the time of sacrifice. FIG. 11D is a graph representing quantitationof Western blots measuring the level of AAT polymeric form in the liversof transgenic animals administered control and AAT-specific iRNAformulations after sacrifice under the experimental protocol set out inFIG. 11A. Units on the y-axis are RDU (relative densiometric units).FIG. 11E shows a Western blot for the polymeric form of AAT in the liverat the time of sacrifice. Control or siLUC=AD-1955 iRNA control, siAATor “drug”=AD-44715 iRNA.

FIGS. 12A-12C depict levels of AAT after a duration study with a singleIV injection of control (Factor VII siRNA; LUC control) or AAT-specific(AD-44715 and AAT_1) iRNAs. iRNAs were administered at a dose of 0.3mg/kg. Animals were sacrificed and liver and serum samples analyzed atday 2 and day 10 for the control-treated group and days 2, 4, 7, 10, and14 for the AAT-specific group. Each bar represent the average of threeanimals. FIG. 12A depicts the level of AAT protein present in the serum(mg/L). FIG. 12B depicts the level of hAAT mRNA in the liver, normalizedto mGAPDH. FIG. 12C depicts the level of mouse Factor VII (mVII) mRNA inthe liver normalized to mouse GAPDH.

FIGS. 13A-13B depict a long term-dosing experiment with AAT siRNA. FIG.13A depicts the experimental protocol with dosing every other week at0.3 mg/kg with either LNP-AAT specific for human AAT or control LNP-Luc.FIG. 13B is a graph of human AAT mRNA levels mouse livers followinglong-term dosing with LNP-AAT or LNP-Luc. Each bar represents theaverage of 6 animals.

FIGS. 14A-14C depict the levels of human AAT in the mice of FIGS.13A-13B. FIG. 14A depicts the results of a western blot withhuman-specific antibody with each lane representing one animal. FIG. 14Ba graph quantitating the level of the monomeric protein (in RDU)detected in FIG. 14A while FIG. 14C is a graph quantitating the level ofthe polymeric protein detected in FIG. 14A.

FIG. 15 depicts the measurement of liver cell injury in the mice ofFIGS. 13A-13B. The graph displays the percentage of hepatocytes whichincorporated BrdU in the livers of mice treated with either LNP-AAT orLNP-Luc. Each bar represents the average of 3 animals.

FIG. 16 depicts level of Col3a1 mRNA in the livers of the mice of FIGS.13A-13B. The mice treated with either LNP-AAT or LNP-Luc areheterozygotes. The graph also displays the level of collagen found inthe livers of both wt and PiZ parents.

FIG. 17 depicts electron micrograph images of liver cells of the mice ofFIGS. 13A-13B. The left micrograph image depicts a cell of a mousetreated with LNP-Luc while the right micrograph depicts a cell of amouse treated by LNP-AAT.

DETAILED DESCRIPTION

Described herein are iRNAs and methods of using them for inhibiting theexpression of a Serpina1 gene in a cell or a mammal where the iRNAtargets a Serpina1 gene. Also provided are compositions and methods fortreating pathological conditions and diseases, such as a liver disorder,in a mammal caused by the expression of a Serpina1 gene. iRNA directsthe sequence-specific degradation of mRNA through a process known as RNAinterference (RNAi).

The iRNAs of the compositions featured herein include an RNA strand (theantisense strand) having a region which is 30 nucleotides or less inlength, i.e., 15-30 nucleotides in length, generally 19-24 nucleotidesin length, which region is substantially complementary to at least partof an mRNA transcript of a Serpina1 gene. The use of these iRNAs enablesthe targeted degradation of mRNAs of genes that are implicated inpathologies associated with Serpina1 expression in mammals. Very lowdosages of Serpina1 iRNAs in particular can specifically and efficientlymediate RNAi, resulting in significant inhibition of expression of aSerpina1 gene. Using cell-based assays, the present inventors havedemonstrated that iRNAs targeting Serpina1 can mediate RNAi, resultingin significant inhibition of expression of a Serpina1 gene. Thus,methods and compositions including these iRNAs are useful for treatingpathological processes caused by accumulation of Serpina1 protein, suchas alpha-1 anti-trypsin liver disease. The following detaileddescription discloses how to make and use compositions containing iRNAsto inhibit the expression of a Serpina1 gene, as well as compositionsand methods for treating diseases and disorders caused by the expressionof this gene. Embodiments of the pharmaceutical compositions featured inthe technology described herein include an iRNA having an antisensestrand comprising a region which is 30 nucleotides or less in length,generally 19-24 nucleotides in length, which region is substantiallycomplementary to at least part of an RNA transcript of a Serpina1 gene,together with a pharmaceutically acceptable carrier. Embodiments ofcompositions featured in the technology described herein also include aniRNA having an antisense strand having a region of complementarity whichis 30 nucleotides or less in length, generally 19-24 nucleotides inlength, and is substantially complementary to at least part of an RNAtranscript of a Serpina1 gene.

Accordingly, in some aspects, pharmaceutical compositions containing aSerpina1 iRNA and a pharmaceutically acceptable carrier, methods ofusing the compositions to inhibit expression of a Serpina1 gene, andmethods of using the pharmaceutical compositions to treat diseasescaused by expression of a Serpina1 gene are featured in the technologydescribed herein.

Liver disease related to Serpina1 deficiency results not strictly from alack of the wildtype protein in the liver, but rather, from anaccumulation of misfolded Serpina1 in the liver, which interferes withhepatic function, leading to chronic liver disease. It can be useful totarget the mutant or variant Serpina1 trancripts in individuals toprevent the accumulation of misfolded protein. In addition, withoutwishing to be bound by theory, it is believed that a therapeutic benefitcan also be provided by inhibiting the expression of the wildtype genein individuals affected by the disease. Without wishing to be bound bytheory, it is thought that reducing expression of wildtype protein, withor without selective reduction in mutant or variant expression, caninfluence the accumulation of misfolded Serpina1 protein. Thus, while itcan, in some instances, be difficult to selectively target a mutanttranscript, a therapeutic benefit can be gained for individuals with, orat risk of, alpha-1 anti-trypsin related liver disease via iRNA mediatedinhibition of wildtype and/or mutant Serpina1 expression.

It is also noted that where delivery of iRNA formulations to the liveris relatively straightforward and selective, administration of an iRNAthat targets wildtype Serpina1 would be expected to exert its primaryeffect in the liver.

Definitions

For convenience, the meaning of certain terms and phrases used in thespecification, examples, and appended claims, are provided below. Ifthere is an apparent discrepancy between the usage of a term in otherparts of this specification and its definition provided in this section,the definition in this section shall prevail.

“G,” “C,” “A,” “T” and “U” each generally stand for a nucleotide thatcontains guanine, cytosine, adenine, thymidine and uracil as a base,respectively. However, it will be understood that the term“ribonucleotide” or “nucleotide” can also refer to a modifiednucleotide, as further detailed below, or a surrogate replacementmoiety. The skilled person is well aware that guanine, cytosine,adenine, and uracil can be replaced by other moieties withoutsubstantially altering the base pairing properties of an oligonucleotidecomprising a nucleotide bearing such replacement moiety. For example,without limitation, a nucleotide comprising inosine as its base can basepair with nucleotides containing adenine, cytosine, or uracil. Hence,nucleotides containing uracil, guanine, or adenine can be replaced inthe nucleotide sequences of dsRNA featured in the technology describedherein by a nucleotide containing, for example, inosine. In anotherexample, adenine and cytosine anywhere in the oligonucleotide can bereplaced with guanine and uracil, respectively to form G-U Wobble basepairing with the target mRNA. Sequences containing such replacementmoieties are suitable for the compositions and methods featured in thetechnology described herein.

As used herein, “Serpina1” refers to a particular polypeptide expressedin a cell. Serpina1 is also known as α-1 antitrypsin or AAT. Thesequence of a human Serpina1 mRNA transcript can be found at NM_000295.4(SEQ ID NO: 01), NM_001002235.2 (SEQ ID NO: 02), NM_001002236.2 (SEQ IDNO: 03), NM_001127700.1 (SEQ ID NO: 04), NM_001127701.1 (SEQ ID NO: 05),NM_001127702.1 (SEQ ID NO: 06), NM_001127703.1 (SEQ ID NO: 07),NM_001127704.1 (SEQ ID NO: 08), NM_001127705.1 (SEQ ID NO: 09),NM_001127706.1 (SEQ ID NO: 10), and/or NM_001127707.1 (SEQ ID NO: 11).The sequence of rhesus Serpina1 mRNA can be found at XM_001099255.2 (SEQID NO: 12) and/or XM_001099044.2 (SEQ ID NO: 13). Over 80 alleles havebeen identified and the “M” allele is considered the wildtype or“normal” allele.

As used herein, “Z-AAT” referes to a mutant allele of Serpina1 in whichthe 342^(nd) amino acid of the protein is changed from a glutamine to alysine as a result of the relevant codon being changed from GAG to AAG.A subject homozygous for a Z allele can be referred to as “PIZZ.” Z-AATmutations account for 95% of Serpina1 deficiency patients and isestimated to be present in 100,000 Americans and ˜3 million individualsworldwide. Z-AAT does not fold correctly in the endoplasmic reticulum,leading to loop-sheet polymers which aggregate and reduce secretion,elicitation of the unfolded protein response, apoptosis, endoplasmicreticulum overload response, autophagy, mitochondrial stress, andaltered hepatocyte function.

As used herein, the term “iRNA” refers to an agent that contains RNA asthat term is defined herein, and which mediates the targeted cleavage ofan RNA transcript via an RNA-induced silencing complex (RISC) pathway.In one embodiment, an iRNA as described herein effects inhibition ofSerpina1 expression. Alternatively, in another embodiment, an iRNA asdescribed herein activates Serpina1 expression.

As used herein, “target sequence” refers to a contiguous portion of thenucleotide sequence of an mRNA molecule formed during the transcriptionof a Serpina1 gene, including mRNA that is a product of RNA processingof a primary transcription product. The target portion of the sequencewill be at least long enough to serve as a substrate for iRNA-directedcleavage at or near that portion. For example, the target sequence willgenerally be from 9-36 nucleotides in length, e.g., 15-30 nucleotides inlength, including all sub-ranges there between. As non-limitingexamples, the target sequence can be from 15-30 nucleotides, 15-26nucleotides, 15-23 nucleotides, 15-22 nucleotides, 15-21 nucleotides,15-20 nucleotides, 15-19 nucleotides, 15-18 nucleotides, 15-17nucleotides, 18-30 nucleotides, 18-26 nucleotides, 18-23 nucleotides,18-22 nucleotides, 18-21 nucleotides, 18-20 nucleotides, 19-30nucleotides, 19-26 nucleotides, 19-23 nucleotides, 19-22 nucleotides,19-21 nucleotides, 19-20 nucleotides, 20-30 nucleotides, 20-26nucleotides, 20-25 nucleotides, 20-24 nucleotides, 20-23 nucleotides,20-22 nucleotides, 20-21 nucleotides, 21-30 nucleotides, 21-26nucleotides, 21-25 nucleotides, 21-24 nucleotides, 21-23 nucleotides, or21-22 nucleotides.

As used herein, the term “strand comprising a sequence” refers to anoligonucleotide comprising a chain of nucleotides that is described bythe sequence referred to using the standard nucleotide nomenclature.

As used herein, and unless otherwise indicated, the term“complementary,” when used to describe a first nucleotide sequence inrelation to a second nucleotide sequence, refers to the ability of anoligonucleotide or polynucleotide comprising the first nucleotidesequence to hybridize and form a duplex structure under certainconditions with an oligonucleotide or polynucleotide comprising thesecond nucleotide sequence, as will be understood by the skilled person.Such conditions can, for example, be stringent conditions, wherestringent conditions can include: 400 mM NaCl, 40 mM PIPES pH 6.4, 1 mMEDTA, 50° C. or 70° C. for 12-16 hours followed by washing. Otherconditions, such as physiologically relevant conditions as can beencountered inside an organism, can apply. The skilled person will beable to determine the set of conditions most appropriate for a test ofcomplementarity of two sequences in accordance with the ultimateapplication of the hybridized nucleotides.

Complementary sequences within an iRNA, e.g., within a dsRNA asdescribed herein, include base-pairing of the oligonucleotide orpolynucleotide comprising a first nucleotide sequence to anoligonucleotide or polynucleotide comprising a second nucleotidesequence over the entire length of one or both nucleotide sequences.Such sequences can be referred to as “fully complementary” with respectto each other herein. However, where a first sequence is referred to as“substantially complementary” with respect to a second sequence herein,the two sequences can be fully complementary, or they can form one ormore, but generally not more than 5, 4, 3 or 2 mismatched base pairsupon hybridization for a duplex up to 30 base pairs, while retaining theability to hybridize under the conditions most relevant to theirultimate application, e.g., inhibition of gene expression via a RISCpathway. However, where two oligonucleotides are designed to form, uponhybridization, one or more single stranded overhangs, such overhangsshall not be regarded as mismatches with regard to the determination ofcomplementarity. For example, a dsRNA comprising one oligonucleotide 21nucleotides in length and another oligonucleotide 23 nucleotides inlength, wherein the longer oligonucleotide comprises a sequence of 21nucleotides that is fully complementary to the shorter oligonucleotide,can yet be referred to as “fully complementary” for the purposesdescribed herein.

“Complementary” sequences, as used herein, can also include, or beformed entirely from, non-Watson-Crick base pairs and/or base pairsformed from non-natural and modified nucleotides, in so far as the aboverequirements with respect to their ability to hybridize are fulfilled.Such non-Watson-Crick base pairs include, but are not limited to, G:UWobble or Hoogstein base pairing.

The terms “complementary,” “fully complementary” and “substantiallycomplementary” herein can be used with respect to the base matchingbetween the sense strand and the antisense strand of a dsRNA, or betweenthe antisense strand of an iRNA agent and a target sequence, as will beunderstood from the context of their use.

As used herein, a polynucleotide that is “substantially complementary toat least part of” a messenger RNA (mRNA) refers to a polynucleotide thatis substantially complementary to a contiguous portion of the mRNA ofinterest (e.g., an mRNA encoding Serpina1). For example, apolynucleotide is complementary to at least a part of a Serpina1 mRNA ifthe sequence is substantially complementary to a non-interrupted portionof an mRNA encoding Serpina1.

The term “double-stranded RNA” or “dsRNA,” as used herein, refers to aniRNA that includes an RNA molecule or complex of molecules having ahybridized duplex region that comprises two anti-parallel andsubstantially complementary nucleic acid strands, which will be referredto as having “sense” and “antisense” orientations with respect to atarget RNA. The duplex region can be of any length that permits specificdegradation of a desired target RNA through a RISC pathway, but willtypically range from 9 to 36 base pairs in length, e.g., 15-30 basepairs in length. Considering a duplex between 9 and 36 base pairs, theduplex can be any length in this range, for example, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31,32, 33, 34, 35, or 36 and any sub-range there between, including, butnot limited to 15-30 base pairs, 15-26 base pairs, 15-23 base pairs,15-22 base pairs, 15-21 base pairs, 15-20 base pairs, 15-19 base pairs,15-18 base pairs, 15-17 base pairs, 18-30 base pairs, 18-26 base pairs,18-23 base pairs, 18-22 base pairs, 18-21 base pairs, 18-20 base pairs,19-30 base pairs, 19-26 base pairs, 19-23 base pairs, 19-22 base pairs,19-21 base pairs, 19-20 base pairs, 20-30 base pairs, 20-26 base pairs,20-25 base pairs, 20-24 base pairs, 20-23 base pairs, 20-22 base pairs,20-21 base pairs, 21-30 base pairs, 21-26 base pairs, 21-25 base pairs,21-24 base pairs, 21-23 base pairs, or 21-22 base pairs. dsRNAsgenerated in the cell by processing with Dicer and similar enzymes aregenerally in the range of 19-22 base pairs in length. One strand of theduplex region of a dsDNA comprises a sequence that is substantiallycomplementary to a region of a target RNA. The two strands forming theduplex structure can be from a single RNA molecule having at least oneself-complementary region, or can be formed from two or more separateRNA molecules. Where the duplex region is formed from two strands of asingle molecule, the molecule can have a duplex region separated by asingle stranded chain of nucleotides (herein referred to as a “hairpinloop”) between the 3′-end of one strand and the 5′-end of the respectiveother strand forming the duplex structure. The hairpin loop can compriseat least one unpaired nucleotide; in some embodiments the hairpin loopcan comprise at least 3, at least 4, at least 5, at least 6, at least 7,at least 8, at least 9, at least 10, at least 20, at least 23 or moreunpaired nucleotides. Where the two substantially complementary strandsof a dsRNA are comprised by separate RNA molecules, those molecules neednot, but can be covalently connected. Where the two strands areconnected covalently by means other than a hairpin loop, the connectingstructure is referred to as a “linker.” The term “siRNA” is also usedherein to refer to a dsRNA as described above.

The skilled artisan will recognize that the term “RNA molecule” or“ribonucleic acid molecule” encompasses not only RNA molecules asexpressed or found in nature, but also analogs and derivatives of RNAcomprising one or more ribonucleotide/ribonucleoside analogs orderivatives as described herein or as known in the art. Strictlyspeaking, a “ribonucleoside” includes a nucleoside base and a ribosesugar, and a “ribonucleotide” is a ribonucleoside with one, two or threephosphate moieties. However, the terms “ribonucleoside” and“ribonucleotide” can be considered to be equivalent as used herein. TheRNA can be modified in the nucleobase structure or in theribose-phosphate backbone structure, e.g., as described herein below.However, the molecules comprising ribonucleoside analogs or derivativesmust retain the ability to form a duplex. As non-limiting examples, anRNA molecule can also include at least one modified ribonucleosideincluding but not limited to a 2′-O-methyl modified nucleoside, anucleoside comprising a 5′ phosphorothioate group, a terminal nucleosidelinked to a cholesteryl derivative or dodecanoic acid bisdecylamidegroup, a locked nucleoside, an abasic nucleoside, a 2′-deoxy-2′-fluoromodified nucleoside, a 2′-amino-modified nucleoside, 2′-alkyl-modifiednucleoside, morpholino nucleoside, a phosphoramidate or a non-naturalbase comprising nucleoside, or any combination thereof. Alternatively,an RNA molecule can comprise at least two modified ribonucleosides, atleast 3, at least 4, at least 5, at least 6, at least 7, at least 8, atleast 9, at least 10, at least 15, at least 20 or more, up to the entirelength of the dsRNA molecule. The modifications need not be the same foreach of such a plurality of modified ribonucleosides in an RNA molecule.In one embodiment, modified RNAs contemplated for use in methods andcompositions described herein are peptide nucleic acids (PNAs) that havethe ability to form the required duplex structure and that permit ormediate the specific degradation of a target RNA via a RISC pathway.

In one aspect, a modified ribonucleoside includes a deoxyribonucleoside.For example, an iRNA agent can comprise one or more deoxynucleosides,including, for example, a deoxynucleoside overhang(s), or one or moredeoxynucleosides within the double stranded portion of a dsRNA. However,it is self evident that under no circumstances is a double stranded DNAmolecule encompassed by the term “iRNA.”

In one aspect, an RNA interference agent includes a single stranded RNAthat interacts with a target RNA sequence to direct the cleavage of thetarget RNA. Without wishing to be bound by theory, long double strandedRNA introduced into plants and invertebrate cells is broken down intosiRNA by a Type III endonuclease known as Dicer (Sharp et al., GenesDev. 2001, 15:485). Dicer, a ribonuclease-III-like enzyme, processes thedsRNA into 19-23 base pair short interfering RNAs with characteristictwo base 3′ overhangs (Bernstein, et al., (2001) Nature 409:363). ThesiRNAs are then incorporated into an RNA-induced silencing complex(RISC) where one or more helicases unwind the siRNA duplex, enabling thecomplementary antisense strand to guide target recognition (Nykanen, etal., (2001) Cell 107:309). Upon binding to the appropriate target mRNA,one or more endonucleases within the RISC cleaves the target to inducesilencing (Elbashir, et al., (2001) Genes Dev. 15:188). Thus, in oneaspect the technology described herein relates to a single stranded RNAthat promotes the formation of a RISC complex to effect silencing of thetarget gene.

As used herein, the term “nucleotide overhang” refers to at least oneunpaired nucleotide that protrudes from the duplex structure of an iRNA,e.g., a dsRNA. For example, when a 3′-end of one strand of a dsRNAextends beyond the 5′-end of the other strand, or vice versa, there is anucleotide overhang. A dsRNA can comprise an overhang of at least onenucleotide; alternatively the overhang can comprise at least twonucleotides, at least three nucleotides, at least four nucleotides, atleast five nucleotides or more. A nucleotide overhang can comprise orconsist of a nucleotide/nucleoside analog, including adeoxynucleotide/nucleoside. The overhang(s) can be on the sense strand,the antisense strand or any combination thereof. Furthermore, thenucleotide(s) of an overhang can be present on the 5′ end, 3′ end orboth ends of either an antisense or sense strand of a dsRNA.

In another aspect, the agent is a single-stranded antisense RNAmolecule. The antisense RNA molecule can have 15-30 nucleotidescomplementary to the target. For example, the antisense RNA molecule hasa sequence of at least 15, 16, 17, 18, 19, 20, or more contiguousnucleotides from one of the antisense sequences of Tables 3 and/or 4.

In one embodiment, the antisense strand of a dsRNA has a 1-10 nucleotideoverhang at the 3′ end and/or the 5′ end. In one embodiment, the sensestrand of a dsRNA has a 1-10 nucleotide overhang at the 3′ end and/orthe 5′ end. In another embodiment, one or more of the nucleotides in theoverhang is replaced with a nucleoside thiophosphate.

The terms “blunt” or “blunt ended” as used herein in reference to adsRNA mean that there are no unpaired nucleotides or nucleotide analogsat a given terminal end of a dsRNA, i.e., no nucleotide overhang. One orboth ends of a dsRNA can be blunt. Where both ends of a dsRNA are blunt,the dsRNA is said to be blunt ended. To be clear, a “blunt ended” dsRNAis a dsRNA that is blunt at both ends, i.e., no nucleotide overhang ateither end of the molecule. Most often such a molecule will bedouble-stranded over its entire length.

The term “antisense strand” or “guide strand” refers to the strand of aniRNA, e.g., a dsRNA, which includes a region that is substantiallycomplementary to a target sequence. As used herein, the term “region ofcomplementarity” refers to the region on the antisense strand that issubstantially complementary to a sequence, for example a targetsequence, as defined herein. Where the region of complementarity is notfully complementary to the target sequence, the mismatches can be in theinternal or terminal regions of the molecule. Generally, the mosttolerated mismatches are in the terminal regions, e.g., within 5, 4, 3,or 2 nucleotides of the 5′ and/or 3′ terminus.

The term “sense strand,” or “passenger strand” as used herein, refers tothe strand of an iRNA that includes a region that is substantiallycomplementary to a region of the antisense strand as that term isdefined herein.

As used herein, in one embodiment, the term “SNALP” refers to a stablenucleic acid-lipid particle. A SNALP represents a vesicle of lipidscoating a reduced aqueous interior comprising a nucleic acid such as aniRNA or a plasmid from which an iRNA is transcribed. SNALPs aredescribed, e.g., in U.S. Patent Application Publication Nos.20060240093, 20070135372, and in International Application No. WO2009082817. These applications are incorporated herein by reference intheir entirety. Examples of “SNALP” formulations are described elsewhereherein.

“Introducing into a cell,” when referring to an iRNA, means facilitatingor effecting uptake or absorption into the cell, as is understood bythose skilled in the art. Absorption or uptake of an iRNA can occurthrough unaided diffusive or active cellular processes, or by auxiliaryagents or devices. The meaning of this term is not limited to cells invitro; an iRNA can also be “introduced into a cell,” wherein the cell ispart of a living organism. In such an instance, introduction into thecell will include the delivery to the organism. For example, for in vivodelivery, iRNA can be injected into a tissue site or administeredsystemically. In vivo delivery can also be by a beta-glucan deliverysystem, such as those described in U.S. Pat. Nos. 5,032,401 and5,607,677, and U.S. Publication No. 2005/0281781 which are herebyincorporated by reference in their entirety. In vitro introduction intoa cell includes methods known in the art such as electroporation andlipofection. Further approaches are described herein below or are knownin the art.

As used herein, the term “inhibit the expression of,” refers to at anleast partial reduction of Serpina1 gene expression in a cell treatedwith an iRNA composition as described herein compared to the expressionof Serpina1 in an untreated cell.

The terms “silence,” “inhibit the expression of,” “down-regulate theexpression of,” “suppress the expression of,” and the like, in so far asthey refer to a Serpina1 gene, herein refer to the at least partialsuppression of the expression of a Serpina1 gene, as manifested by areduction of the amount of Serpina1 mRNA which can be isolated from ordetected in a first cell or group of cells in which a Serpina1 gene istranscribed and which has or have been treated such that the expressionof a Serpina1 gene is inhibited, as compared to a second cell or groupof cells substantially identical to the first cell or group of cells butwhich has or have not been so treated (control cells). The degree ofinhibition is usually expressed in terms of

${\frac{\left( {{mRNA}\mspace{14mu} {in}\mspace{14mu} {control}\mspace{14mu} {cells}} \right) - \left( {{mRNA}\mspace{14mu} {in}\mspace{14mu} {treated}\mspace{14mu} {cells}} \right)}{\left( {{mRNA}\mspace{14mu} {in}\mspace{14mu} {control}\mspace{14mu} {cells}} \right)} \cdot 100}\%$

Alternatively, the degree of inhibition can be given in terms of areduction of a parameter that is functionally linked to Serpina1 geneexpression, e.g., the amount of protein encoded by a Serpina1 gene, orthe number of cells displaying a certain phenotype, e.g., alteredhepatocyte function. In principle, Serpina1 gene silencing can bedetermined in any cell expressing Serpina1, either constitutively or bygenomic engineering, and by any appropriate assay. However, when areference is needed in order to determine whether a given iRNA inhibitsthe expression of the Serpina1 gene by a certain degree and therefore isencompassed by the instant invention, the assays provided in theExamples below shall serve as such reference.

For example, in certain instances, expression of a Serpina1 gene issuppressed by at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or50% by administration of an iRNA featured herein. In some embodiments, aSerpina1 gene is suppressed by at least about 60%, 70%, or 80% byadministration of an iRNA featured herein. In some embodiments, aSerpina1gene is suppressed by at least about 85%, 90%, 95%, 98%, 99% ormore by administration of an iRNA as described herein.

As used herein in the context of Serpina1 expression, the terms “treat,”“treatment,” and the like, refer to relief from or alleviation ofpathological processes mediated by Serpina1 expression. In someembodiments, the terms “treat,” “treatment,” and the like mean torelieve or alleviate at least one symptom associated with suchcondition, or to slow or reverse the progression or anticipatedprogression of such condition, such as slowing the progression of aliver disorder, such as liver inflammation or cirrhosis.

By “lower” in the context of a disease marker or symptom is meant astatistically significant decrease in such level. The decrease can be,for example, at least 10%, at least 20%, at least 30%, at least 40% ormore, and is preferably down to a level accepted as within the range ofnormal for an individual without such disorder.

As used herein, “prevention” or “preventing,” when used in reference toa disease, disorder or symptoms thereof, refers to a reduction in thelikelihood that an individual will develop a disease of disorder, e.g.,a respiratory disorder. The likelihood of developing a disease ordisorder is reduced, for example, when an individual having one or morerisk factors for a disease or disorder either fails to develop thedisorder or develops such disease or disorder at a later time or withless severity, statistically speaking, relative to a population havingthe same risk factors and not receiving treatment as described herein.The failure to develop symptoms of a disease, or the development ofreduced (e.g., by at least 10% on a clinically accepted scale for thatdisease or disorder) or delayed (e.g., by days, weeks, months or years)symptoms is considered effective prevention.

As used herein, the phrases “therapeutically effective amount” and“prophylactically effective amount” refer to an amount that provides atherapeutic benefit in the treatment, prevention, or management ofpathological processes mediated by Serpina1 expression or an overtsymptom of pathological processes mediated by Serpina1 expression. Thespecific amount that is therapeutically effective can be readilydetermined by an ordinary medical practitioner, and can vary dependingon factors known in the art, such as, for example, the type ofpathological processes mediated by Serpina1 expression, the patient'shistory and age, the stage of pathological processes mediated bySerpina1 expression, and the administration of other agents that inhibitpathological processes mediated by Serpina1 expression.

As used herein, a “pharmaceutical composition” comprises apharmacologically effective amount of an iRNA and a pharmaceuticallyacceptable carrier. As used herein, “pharmacologically effectiveamount,” “therapeutically effective amount” or simply “effective amount”refers to that amount of an iRNA effective to produce the intendedpharmacological, therapeutic or preventive result. For example, if agiven clinical treatment is considered effective when there is at leasta 10% reduction in a measurable parameter associated with a disease ordisorder, a therapeutically effective amount of a drug for the treatmentof that disease or disorder is the amount necessary to effect at least a10% reduction in that parameter. For example, a therapeuticallyeffective amount of an iRNA targeting Serpina1 can reduce Serpina1protein levels by at least 10%.

As used herein, the term “alpha-1 anti-trypsin liver disease” refers toa condition in which Serpina1 protein accumulates in the liver,resulting in liver injury and inflammation. Alpha-1 anti-trypsin liverdisease can be hereditary and is known to occur more often in subjectswith one or more copies of certain alleles (e.g. the PiZ, PiS, orPiM(Malton) alleles). Without wishing to be bound by theory, it isthought that alleles associated with a greater risk of developingalpha-1 anti-trypsin liver disease encode forms of Serpina1 which aresubject to misfolding and are not properly secreted from thehepatocytes. The cellular responses to these misfolded proteins caninclude the unfolded protein response, ERAD, apoptosis, ER overloadresponse, autophagy, mitochondrial stress and altered hepatocytefunction. The injuries to the hepatocytes can lead to symptoms such as,but not limited to, inflammation, cholestasis, fibrosis, cirrhosis,prolonged obstructive jaundice, increased transaminases, portalhypertension and/or hepatocellular carcinoma. Without wishing to bebound by theory, the highly variable clinical course of this disease issuggestive of modifiers or “second hits” as contributors to developingsymptoms or progressing in severity. For example, subjects with a PiZallele can be more sensitive to Hepatitis C infections or alcohol abuseand more likely to develop a liver disorder if exposed to such factors.A deficiency of alpha-1 antitrypsin can also cause or contribute to thedevelopment of early onset emphysema, necrotizing panniculitis,bronchiectasis, and/or prolonged neonatal jaundice. Some patients havingor at risk of having a deficiency of alpha-1 antitrypsin are identifiedby screening when they have family members affected by an alpha-1antitrypsin deficiency.

As used herein, the terms “liver disease” and “liver disorder” are usedinterchangeably and refer to any disorder which impairs liver function.A liver disorder can arise from one or more sources, which include butare not limited to, accumulation of Serpina1 protein in the liver and/orliver cells, viral infections, parasitic infections, geneticpredisposition, autoimmune diseases, exposure to radiation, exposure tohepatotoxic compounds, mechanical injuries, various environmentaltoxins, alcohol, acetaminophen, a combination of alcohol andacetaminophen, inhalation anesthetics, niacin, chemotherapeutics,antibiotics, analgesics, antiemetics and the herbal supplement kava.Symptoms of a liver disorder include, but are not limited to,inflammation, cirrhosis, fibrosis, hepatocellular carcinoma, necrosis,steatosis, fibrosis, cholestatis and/or reduction and/or loss ofhepatocyte function. Liver disorders often lead to cirrhosis. Cirrhosisis a pathological condition associated with chronic liver damage thatincludes extensive fibrosis and regenerative nodules. “Fibrosis” as usedherein refers to the proliferation of fibroblasts and the formation ofscar tissue in the liver.

The phrase “liver function” refers to one or more of the manyphysiological functions performed by the liver. Such functions include,but are not limited to, regulating blood sugar levels, endocrineregulation, enzyme systems, interconversion of metabolites (e.g., ketonebodies, sterols and steroids and amino acids); manufacturing bloodproteins such as fibrinogen, serum albumin, and cholinesterase,erythropoietic function, detoxification, bile formation, and vitaminstorage. Several tests to examine liver function are known in the art,including, for example, measuring alanine amino transferase (ALT),alkaline phosphatase, bilirubin, prothrombin, and albumin.

The term “pharmaceutically acceptable carrier” refers to a carrier foradministration of a therapeutic agent. Such carriers include, but arenot limited to, saline, buffered saline, dextrose, water, glycerol,ethanol, and combinations thereof. The term specifically excludes cellculture medium. For drugs administered orally, pharmaceuticallyacceptable carriers include, but are not limited to pharmaceuticallyacceptable excipients such as inert diluents, disintegrating agents,binding agents, lubricating agents, sweetening agents, flavoring agents,coloring agents and preservatives. Suitable inert diluents includesodium and calcium carbonate, sodium and calcium phosphate, and lactose,while corn starch and alginic acid are suitable disintegrating agents.Binding agents can include starch and gelatin, while the lubricatingagent, if present, will generally be magnesium stearate, stearic acid ortalc. If desired, the tablets can be coated with a material such asglyceryl monostearate or glyceryl distearate, to delay absorption in thegastrointestinal tract. Agents included in drug formulations aredescribed further herein below.

As used herein, a therapeutic that “prevents” a liver disease is acomposition that, in a statistical sample, reduces the occurrence ofliver dieases or symptoms of liver disease in the treated samplerelative to an untreated control sample, or delays the onset or reducesthe severity of one or more symptoms of the disorder or conditionrelative to the untreated control sample.

As used herein, a “subject” is a mammal, e. g. a dog, horse, cat, andother non-human primates. In a preferred embodiment, a subject is ahuman.

As used herein, the term “LNPXX”, wherein the “XX” are numerals, is alsoreferred to as “AFXX” herein. For example, LNP09 is also referred toAF09 and LNP12 is also known as or referred to as AF12.

As used herein, the term “comprising” or “comprises” is used inreference to compositions, methods, and respective component(s) thereof,that are essential to the invention, yet open to the inclusion ofunspecified elements, whether essential or not.

As used herein, the term “consisting essentially of” refers to thoseelements required for a given embodiment. The term permits the presenceof elements that do not materially affect the basic and novel orfunctional characteristic(s) of that embodiment of the invention.

The term “consisting of” refers to compositions, methods, and respectivecomponents thereof as described herein, which are exclusive of anyelement not recited in that description of the embodiment.

Double-Stranded Ribonucleic Acid (dsRNA)

Described herein are iRNA agents that inhibit the expression of theSerpina1 gene. In one embodiment, the iRNA agent includesdouble-stranded ribonucleic acid (dsRNA) molecules for inhibiting theexpression of a Serpina1 gene in a cell or mammal, e.g., in a humanhaving a lipid disorder, where the dsRNA includes an antisense strandhaving a region of complementarity which is complementary to at least apart of an mRNA formed in the expression of a Serpina1 gene, and wherethe region of complementarity is 30 nucleotides or less in length,generally 19-24 nucleotides in length, and where the dsRNA, upon contactwith a cell expressing the Serpina1 gene, inhibits the expression of theSerpina1 gene by at least 10% as assayed by, for example, a PCR orbranched DNA (bDNA)-based method, or by a protein-based method, such asby Western blot. In one embodiment, the iRNA agent activates theexpression of a Serpina1 gene in a cell or mammal. Expression of aSerpina1 gene in cell culture, such as in COS cells, HeLa cells, primaryhepatocytes, HepG2 cells, primary cultured cells or in a biologicalsample from a subject, can be assayed by measuring Serpina1 mRNA levels,such as by bDNA or TaqMan assay, or by measuring protein levels, such asby immunofluorescence analysis, using, for example, Western Blotting orflowcytometric techniques

A dsRNA includes two RNA strands that are complementary and hybridize toform a duplex structure under conditions in which the dsRNA will beused. One strand of a dsRNA (the antisense strand) includes a region ofcomplementarity that is substantially complementary, and generally fullycomplementary, to a target sequence. The target sequence can be derivedfrom the sequence of an mRNA formed during the expression of a Serpina1gene. The other strand (the sense strand) includes a region that iscomplementary to the antisense strand, such that the two strandshybridize and form a duplex structure when combined under suitableconditions. Generally, the duplex structure is between 15 and 30inclusive, more generally between 18 and 25 inclusive, yet moregenerally between 19 and 24 inclusive, and most generally between 19 and21 base pairs in length, inclusive. Similarly, the region ofcomplementarity to the target sequence is between 15 and 30 inclusive,more generally between 18 and 25 inclusive, yet more generally between19 and 24 inclusive, and most generally between 19 and 21 nucleotides inlength, inclusive. In some embodiments, the dsRNA is between 15 and 20nucleotides in length, inclusive, and in other embodiments, the dsRNA isbetween 25 and 30 nucleotides in length, inclusive. As the ordinarilyskilled person will recognize, the targeted region of an RNA targetedfor cleavage will most often be part of a larger RNA molecule, often anmRNA molecule. Where relevant, a “part” of an mRNA target is acontiguous sequence of an mRNA target of sufficient length to be asubstrate for RNAi-directed cleavage (i.e., cleavage through a RISCpathway). dsRNAs having duplexes as short as 9 base pairs can, undersome circumstances, mediate RNAi-directed RNA cleavage. Most often atarget will be at least 15 nucleotides in length, preferably 15-30nucleotides in length.

One of skill in the art will also recognize that the duplex region is aprimary functional portion of a dsRNA, e.g., a duplex region of 9 to 36,e.g., 15-30 base pairs. Thus, in one embodiment, to the extent that itbecomes processed to a functional duplex of e.g., 15-30 base pairs thattargets a desired RNA for cleavage, an RNA molecule or complex of RNAmolecules having a duplex region greater than 30 base pairs is a dsRNA.Thus, an ordinarily skilled artisan will recognize that in oneembodiment, then, a miRNA is a dsRNA. In another embodiment, a dsRNA isnot a naturally occurring miRNA. In another embodiment, an iRNA agentuseful to target Serpina1 expression is not generated in the target cellby cleavage of a larger dsRNA.

A dsRNA as described herein can further include one or moresingle-stranded nucleotide overhangs. The dsRNA can be synthesized bystandard methods known in the art as further discussed below, e.g., byuse of an automated DNA synthesizer, such as are commercially availablefrom, for example, Biosearch, Applied Biosystems, Inc. In oneembodiment, a Serpina1 gene is a human Serpina1 gene. In anotherembodiment, the Serpina1 gene is a rhesus Serpina1 gene. In anotherembodiment the Serpina1 gene is a mouse or a rat Serpina1 gene. Inspecific embodiments, the first sequence is a sense strand of a dsRNAthat includes a sense sequence from one of Tables 3 and 4, and thesecond sequence is selected from the group consisting of the antisensesequences of one of Tables 3 and 4. Alternative dsRNA agents that targetelsewhere in the target sequence provided in Tables 3 and 4 can readilybe determined using the target sequence and the flanking Serpina1sequence.

In one aspect, a dsRNA will include at least two nucleotide sequences, asense and an anti-sense sequence, whereby the sense strand is selectedfrom the groups of sequences provided in Tables 3 and 4, and thecorresponding antisense strand of the sense strand selected from Tables3 and 4. In this aspect, one of the two sequences is complementary tothe other of the two sequences, with one of the sequences beingsubstantially complementary to a sequence of an mRNA generated in theexpression of a Serpina1 gene. As such, in this aspect, a dsRNA willinclude two oligonucleotides, where one oligonucleotide is described asthe sense strand in Tables 3 and 4, and the second oligonucleotide isdescribed as the corresponding antisense strand of the sense strand inTables 3 and 4. As described elsewhere herein and as known in the art,the complementary sequences of a dsRNA can also be contained asself-complementary regions of a single nucleic acid molecule, as opposedto being on separate oligonucleotides.

The skilled person is well aware that dsRNAs having a duplex structureof between 20 and 23, but specifically 21, base pairs have been hailedas particularly effective in inducing RNA interference (Elbashir et al.,EMBO 2001, 20:6877-6888). However, others have found that shorter orlonger RNA duplex structures can be effective as well. In theembodiments described above, by virtue of the nature of theoligonucleotide sequences provided in Tables 3 and 4 dsRNAs describedherein can include at least one strand of a length of minimally 21 nt.It can be reasonably expected that shorter duplexes having one of thesequences of Tables 3 and 4 minus only a few nucleotides on one or bothends can be similarly effective as compared to the dsRNAs describedabove. Hence, dsRNAs having a partial sequence of at least 15, 16, 17,18, 19, 20, or more contiguous nucleotides from one of the sequences ofTables 3 and 4, and differing in their ability to inhibit the expressionof a Serpina1 gene by not more than 5, 10, 15, 20, 25, or 30% inhibitionfrom a dsRNA comprising the full sequence, are contemplated according tothe technology described herein.

In addition, the RNAs provided in Tables 3 and 4 identify a site in aSerpina1 transcript that is susceptible to RISC-mediated cleavage. Assuch, the technology described herein further features iRNAs that targetwithin one of such sequences. As used herein, an iRNA is said to targetwithin a particular site of an RNA transcript if the iRNA promotescleavage of the transcript anywhere within that particular site. Such aniRNA will generally include at least 15 contiguous nucleotides from oneof the sequences provided in Tables 3 and 4 coupled to additionalnucleotide sequences taken from the region contiguous to the selectedsequence in a Serpina1 gene.

While a target sequence is generally 15-30 nucleotides in length, thereis wide variation in the suitability of particular sequences in thisrange for directing cleavage of any given target RNA. Various softwarepackages and the guidelines set out herein provide guidance for theidentification of optimal target sequences for any given gene target,but an empirical approach can also be taken in which a “window” or“mask” of a given size (as a non-limiting example, 21 nucleotides) isliterally or figuratively (including, e.g., in silico) placed on thetarget RNA sequence to identify sequences in the size range that canserve as target sequences. By moving the sequence “window” progressivelyone nucleotide upstream or downstream of an initial target sequencelocation, the next potential target sequence can be identified, untilthe complete set of possible sequences is identified for any giventarget size selected. This process, coupled with systematic synthesisand testing of the identified sequences (using assays as describedherein or as known in the art) to identify those sequences that performoptimally can identify those RNA sequences that, when targeted with aniRNA agent, mediate the best inhibition of target gene expression. Thus,while the sequences identified, for example, in Tables 3 and 4 representeffective target sequences, it is contemplated that further optimizationof inhibition efficiency can be achieved by progressively “walking thewindow” one nucleotide upstream or downstream of the given sequences toidentify sequences with equal or better inhibition characteristics.

Further, it is contemplated that for any sequence identified, e.g., inTables 3 and 4, further optimization could be achieved by systematicallyeither adding or removing nucleotides to generate longer or shortersequences and testing those and sequences generated by walking a windowof the longer or shorter size up or down the target RNA from that point.Again, coupling this approach to generating new candidate targets withtesting for effectiveness of iRNAs based on those target sequences in aninhibition assay as known in the art or as described herein can lead tofurther improvements in the efficiency of inhibition. Further still,such optimized sequences can be adjusted by, e.g., the introduction ofmodified nucleotides as described herein or as known in the art,addition or changes in overhang, or other modifications as known in theart and/or discussed herein to further optimize the molecule (e.g.,increasing serum stability or circulating half-life, increasing thermalstability, enhancing transmembrane delivery, targeting to a particularlocation or cell type, increasing interaction with silencing pathwayenzymes, increasing release from endosomes, etc.) as an expressioninhibitor.

An iRNA as described herein can contain one or more mismatches to thetarget sequence. In one embodiment, an iRNA as described herein containsno more than 3 mismatches. If the antisense strand of the iRNA containsmismatches to a target sequence, it is preferable that the area ofmismatch not be located in the center of the region of complementarity.If the antisense strand of the iRNA contains mismatches to the targetsequence, it is preferable that the mismatch be restricted to be withinthe last 5 nucleotides from either the 5′ or 3′ end of the region ofcomplementarity. For example, for a 23 nucleotide iRNA agent RNA strandwhich is complementary to a region of a Serpina1 gene, the RNA strandgenerally does not contain any mismatch within the central 13nucleotides. The methods described herein or methods known in the artcan be used to determine whether an iRNA containing a mismatch to atarget sequence is effective in inhibiting the expression of a Serpina1gene. Consideration of the efficacy of iRNAs with mismatches ininhibiting expression of a Serpina1 gene is important, especially if theparticular region of complementarity in a Serpina1 gene is known to havepolymorphic sequence variation within the population.

In one embodiment, at least one end of a dsRNA has a single-strandednucleotide overhang of 1 to 4, generally 1 or 2 nucleotides. dsRNAshaving at least one nucleotide overhang can have unexpectedly superiorinhibitory properties relative to their blunt-ended counterparts. In yetanother embodiment, the RNA of an iRNA, e.g., a dsRNA, is chemicallymodified to enhance stability or other beneficial characteristics. Thenucleic acids featured in the technology described herein can besynthesized and/or modified by methods well established in the art, suchas those described in “Current protocols in nucleic acid chemistry,”Beaucage, S. L. et al. (Edrs.), John Wiley & Sons, Inc., New York, N.Y.,USA, which is hereby incorporated herein by reference. Modificationsinclude, for example, (a) end modifications, e.g., 5′ end modifications(phosphorylation, conjugation, inverted linkages, etc.) 3′ endmodifications (conjugation, DNA nucleotides, inverted linkages, etc.),(b) base modifications, e.g., replacement with stabilizing bases,destabilizing bases, or bases that base pair with an expanded repertoireof partners, removal of bases (abasic nucleotides), or conjugated bases,(c) sugar modifications (e.g., at the 2′ position or 4′ position) orreplacement of the sugar, as well as (d) backbone modifications,including modification or replacement of the phosphodiester linkages.Specific examples of RNA compounds useful in the embodiments describedherein include, but are not limited to RNAs containing modifiedbackbones or no natural internucleoside linkages. RNAs having modifiedbackbones include, among others, those that do not have a phosphorusatom in the backbone. For the purposes of this specification, and assometimes referenced in the art, modified RNAs that do not have aphosphorus atom in their internucleoside backbone can also be consideredto be oligonucleosides. In particular embodiments, the modified RNA willhave a phosphorus atom in its internucleoside backbone.

Modified RNA backbones include, for example, phosphorothioates, chiralphosphorothioates, phosphorodithioates, phosphotriesters,aminoalkylphosphotriesters, methyl and other alkyl phosphonatesincluding 3′-alkylene phosphonates and chiral phosphonates,phosphinates, phosphoramidates including 3′-amino phosphoramidate andaminoalkylphosphoramidates, thionophosphoramidates,thionoalkylphosphonates, thionoalkylphosphotriesters, andboranophosphates having normal 3′-5′ linkages, 2′-5′ linked analogs ofthese, and those) having inverted polarity wherein the adjacent pairs ofnucleoside units are linked 3′-5′ to 5′-3′ or 2′-5′ to 5′-2′. Varioussalts, mixed salts and free acid forms are also included.

Representative U.S. patents that teach the preparation of the abovephosphorus-containing linkages include, but are not limited to, U.S.Pat. Nos. 3,687,808; 4,469,863; 4,476,301; 5,023,243; 5,177,195;5,188,897; 5,264,423; 5,276,019; 5,278,302; 5,286,717; 5,321,131;5,399,676; 5,405,939; 5,453,496; 5,455,233; 5,466,677; 5,476,925;5,519,126; 5,536,821; 5,541,316; 5,550,111; 5,563,253; 5,571,799;5,587,361; 5,625,050; 6,028,188; 6,124,445; 6,160,109; 6,169,170;6,172,209; 6,239,265; 6,277,603; 6,326,199; 6,346,614; 6,444,423;6,531,590; 6,534,639; 6,608,035; 6,683,167; 6,858,715; 6,867,294;6,878,805; 7,015,315; 7,041,816; 7,273,933; 7,321,029; and U.S. Pat.RE39464, each of which is herein incorporated by reference

Modified RNA backbones that do not include a phosphorus atom thereinhave backbones that are formed by short chain alkyl or cycloalkylinternucleoside linkages, mixed heteroatoms and alkyl or cycloalkylinternucleoside linkages, or one or more short chain heteroatomic orheterocyclic internucleoside linkages. These include those havingmorpholino linkages (formed in part from the sugar portion of anucleoside); siloxane backbones; sulfide, sulfoxide and sulfonebackbones; formacetyl and thioformacetyl backbones; methylene formacetyland thioformacetyl backbones; alkene containing backbones; sulfamatebackbones; methyleneimino and methylenehydrazino backbones; sulfonateand sulfonamide backbones; amide backbones; and others having mixed N,O, S and CH₂ component parts.

Representative U.S. patents that teach the preparation of the aboveoligonucleosides include, but are not limited to, U.S. Pat. Nos.5,034,506; 5,166,315; 5,185,444; 5,214,134; 5,216,141; 5,235,033;5,64,562; 5,264,564; 5,405,938; 5,434,257; 5,466,677; 5,470,967;5,489,677; 5,541,307; 5,561,225; 5,596,086; 5,602,240; 5,608,046;5,610,289; 5,618,704; 5,623,070; 5,663,312; 5,633,360; 5,677,437; and,5,677,439, each of which is herein incorporated by reference.

In other embodiments, suitable RNA mimetics suitable are contemplatedfor use in iRNAs, in which both the sugar and the internucleosidelinkage, i.e., the backbone, of the nucleotide units are replaced withnovel groups. The base units are maintained for hybridization with anappropriate nucleic acid target compound. One such oligomeric compound,an RNA mimetic that has been shown to have excellent hybridizationproperties, is referred to as a peptide nucleic acid (PNA). In PNAcompounds, the sugar backbone of an RNA is replaced with an amidecontaining backbone, in particular an aminoethylglycine backbone. Thenucleobases are retained and are bound directly or indirectly to azanitrogen atoms of the amide portion of the backbone. Representative U.S.patents that teach the preparation of PNA compounds include, but are notlimited to, U.S. Pat. Nos. 5,539,082; 5,714,331; and 5,719,262, each ofwhich is herein incorporated by reference. Further teaching of PNAcompounds can be found, for example, in Nielsen et al., Science, 1991,254, 1497-1500.

Some embodiments featured in the technology described herein includeRNAs with phosphorothioate backbones and oligonucleosides withheteroatom backbones, and in particular —CH₂—NH—CH₂—,—CH₂—N(CH₃)—O—CH₂—[known as a methylene (methylimino) or MMI backbone],—CH₂—O—N(CH₃)—CH₂—, —CH₂—N(CH₃)—N(CH₃)—CH₂— and —N(CH₃)—CH₂—CH₂—[whereinthe native phosphodiester backbone is represented as —O—P—O—CH₂—] of theabove-referenced U.S. Pat. No. 5,489,677, and the amide backbones of theabove-referenced U.S. Pat. No. 5,602,240. In some embodiments, the RNAsfeatured herein have morpholino backbone structures of theabove-referenced U.S. Pat. No. 5,034,506.

Modified RNAs can also contain one or more substituted sugar moieties.The iRNAs, e.g., dsRNAs, featured herein can include one of thefollowing at the 2′ position: OH; F; O—, S—, or N-alkyl; O—, S—, orN-alkenyl; O—, S- or N-alkynyl; or O-alkyl-O-alkyl, wherein the alkyl,alkenyl and alkynyl can be substituted or unsubstituted C₁ to C₁₀ alkylor C₂ to C₁₀ alkenyl and alkynyl. Exemplary suitable modificationsinclude O[(CH₂)_(n)O]_(m)CH₃, O(CH₂)._(n)OCH₃, O(CH₂)_(n)NH₂,O(CH₂)_(n)CH₃, O(CH₂)_(n)ONH₂, and O(CH₂)_(n)ON[(CH₂)_(n)CH₃)]₂, where nand m are from 1 to about 10. In other embodiments, dsRNAs include oneof the following at the 2′ position: C₁ to C₁₀ lower alkyl, substitutedlower alkyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH₃, OCN,Cl, Br, CN, CF₃, OCF₃, SOCH₃, SO₂CH₃, ONO₂, NO₂, N₃, NH₂,heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino,substituted silyl, an RNA cleaving group, a reporter group, anintercalator, a group for improving the pharmacokinetic properties of aniRNA, or a group for improving the pharmacodynamic properties of aniRNA, and other substituents having similar properties. In someembodiments, the modification includes a 2′-methoxyethoxy(2′-O—CH₂CH₂OCH₃, also known as 2′-O-(2-methoxyethyl) or 2′-MOE) (Martinet al., Helv. Chim. Acta, 1995, 78:486-504) i.e., an alkoxy-alkoxygroup. Another exemplary modification is 2′-dimethylaminooxyethoxy,i.e., a O(CH₂)₂ON(CH₃)₂ group, also known as 2′-DMAOE, as described inexamples herein below, and 2′-dimethylaminoethoxyethoxy (also known inthe art as 2′-O-dimethylaminoethoxyethyl or 2′-DMAEOE), i.e.,2′-O—CH₂—O—CH₂—N(CH₂)₂, also described in examples herein below.

Other modifications include 2′-methoxy (2′-OCH₃), 2′-aminopropoxy(2′-OCH₂CH₂CH₂NH₂) and 2′-fluoro (2′-F). Similar modifications can alsobe made at other positions on the RNA of an iRNA, particularly the 3′position of the sugar on the 3′ terminal nucleotide or in 2′-5′ linkeddsRNAs and the 5′ position of 5′ terminal nucleotide. iRNAs can alsohave sugar mimetics such as cyclobutyl moieties in place of thepentofuranosyl sugar. Representative U.S. patents that teach thepreparation of such modified sugar structures include, but are notlimited to, U.S. Pat. Nos. 4,981,957; 5,118,800; 5,319,080; 5,359,044;5,393,878; 5,446,137; 5,466,786; 5,514,785; 5,519,134; 5,567,811;5,576,427; 5,591,722; 5,597,909; 5,610,300; 5,627,053; 5,639,873;5,646,265; 5,658,873; 5,670,633; and 5,700,920, certain of which arecommonly owned with the instant application, and each of which is hereinincorporated by reference.

An iRNA can also include nucleobase (often referred to in the art simplyas “base”) modifications or substitutions. As used herein, “unmodified”or “natural” nucleobases include the purine bases adenine (A) andguanine (G), and the pyrimidine bases thymine (T), cytosine (C) anduracil (U). Modified nucleobases include other synthetic and naturalnucleobases such as 5-methylcytosine (5-me-C), 5-hydroxymethyl cytosine,xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkylderivatives of adenine and guanine, 2-propyl and other alkyl derivativesof adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine,5-halouracil and cytosine, 5-propynyl uracil and cytosine, 6-azo uracil,cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo,8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl anal other 8-substitutedadenines and guanines, 5-halo, particularly 5-bromo, 5-trifluoromethyland other 5-substituted uracils and cytosines, 7-methylguanine and7-methyladenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and7-daazaadenine and 3-deazaguanine and 3-deazaadenine. Furthernucleobases include those disclosed in U.S. Pat. No. 3,687,808, thosedisclosed in Modified Nucleosides in Biochemistry, Biotechnology andMedicine, Herdewijn, P. ed. Wiley-VCH, 2008; those disclosed in TheConcise Encyclopedia Of Polymer Science And Engineering, pages 858-859,Kroschwitz, J. L, ed. John Wiley & Sons, 1990, these disclosed byEnglisch et al., Angewandte Chemie, International Edition, 1991, 30,613, and those disclosed by Sanghvi, Y S., Chapter 15, dsRNA Researchand Applications, pages 289-302, Crooke, S. T. and Lebleu, B., Ed., CRCPress, 1993. Certain of these nucleobases are particularly useful forincreasing the binding affinity of the oligomeric compounds featured inthe technology described herein. These include 5-substitutedpyrimidines, 6-azapyrimidines and N-2, N-6 and 0-6 substituted purines,including 2-aminopropyladenine, 5-propynyluracil and 5-propynylcytosine.5-methylcytosine substitutions have been shown to increase nucleic acidduplex stability by 0.6-1.2° C. (Sanghvi, Y. S., Crooke, S. T. andLebleu, B., Eds., dsRNA Research and Applications, CRC Press, BocaRaton, 1993, pp. 276-278) and are exemplary base substitutions, evenmore particularly when combined with 2′-O-methoxyethyl sugarmodifications.

Representative U.S. patents that teach the preparation of certain of theabove noted modified nucleobases as well as other modified nucleobasesinclude, but are not limited to, the above noted U.S. Pat. No.3,687,808, as well as U.S. Pat. Nos. 4,845,205; 5,130,30; 5,134,066;5,175,273; 5,367,066; 5,432,272; 5,457,187; 5,459,255; 5,484,908;5,502,177; 5,525,711; 5,552,540; 5,587,469; 5,594,121, 5,596,091;5,614,617; 5,681,941; 6,015,886; 6,147,200; 6,166,197; 6,222,025;6,235,887; 6,380,368; 6,528,640; 6,639,062; 6,617,438; 7,045,610;7,427,672; and 7,495,088, each of which is herein incorporated byreference, and U.S. Pat. No. 5,750,692, also herein incorporated byreference.

The RNA of an iRNA can also be modified to include one or more lockednucleic acids (LNA). A locked nucleic acid is a nucleotide having amodified ribose moiety in which the ribose moiety comprises an extrabridge connecting the 2′ and 4′ carbons. This structure effectively“locks” the ribose in the 3′-endo structural conformation. The additionof locked nucleic acids to siRNAs has been shown to increase siRNAstability in serum, and to reduce off-target effects (Elmen, J. et al.,(2005) Nucleic Acids Research 33(1):439-447; Mook, O R. et al., (2007)Mol Canc Ther 6(3):833-843; Grunweller, A. et al., (2003) Nucleic AcidsResearch 31(12):3185-3193).

Representative U.S. patents that teach the preparation of locked nucleicacid nucleotides include, but are not limited to, the following: U.S.Pat. Nos. 6,268,490; 6,670,461; 6,794,499; 6,998,484; 7,053,207;7,084,125; and 7,399,845, each of which is herein incorporated byreference in its entirety.

Another modification of the RNA of an iRNA featured in the technologydescribed herein involves chemically linking to the RNA one or moreligands, moieties or conjugates that enhance the activity, cellulardistribution or cellular uptake of the iRNA. Such moieties include butare not limited to lipid moieties such as a cholesterol moiety(Letsinger et al., Proc. Natl. Acid. Sci. USA, 1989, 86: 6553-6556),cholic acid (Manoharan et al., Biorg. Med. Chem. Let., 1994,4:1053-1060), a thioether, e.g., beryl-S-tritylthiol (Manoharan et al.,Ann. N.Y. Acad. Sci., 1992, 660:306-309; Manoharan et al., Biorg. Med.Chem. Let., 1993, 3:2765-2770), a thiocholesterol (Oberhauser et al.,Nucl. Acids Res., 1992, 20:533-538), an aliphatic chain, e.g.,dodecandiol or undecyl residues (Saison-Behmoaras et al., EMBO J, 1991,10:1111-1118; Kabanov et al., FEBS Lett., 1990, 259:327-330; Svinarchuket al., Biochimie, 1993, 75:49-54), a phospholipid, e.g.,di-hexadecyl-rac-glycerol or triethylammonium1,2-di-O-hexadecyl-rac-glycero-3-phosphonate (Manoharan et al.,Tetrahedron Lett., 1995, 36:3651-3654; Shea et al., Nucl. Acids Res.,1990, 18:3777-3783), a polyamine or a polyethylene glycol chain(Manoharan et al., Nucleosides & Nucleotides, 1995, 14:969-973), oradamantane acetic acid (Manoharan et al., Tetrahedron Lett., 1995,36:3651-3654), a palmityl moiety (Mishra et al., Biochim. Biophys. Acta,1995, 1264:229-237), or an octadecylamine orhexylamino-carbonyloxycholesterol moiety (Crooke et al., J. Pharmacol.Exp. Ther., 1996, 277:923-937).

In one embodiment, a ligand alters the distribution, targeting orlifetime of an iRNA agent into which it is incorporated. In preferredembodiments a ligand provides an enhanced affinity for a selectedtarget, e.g., molecule, cell or cell type, compartment, e.g., a cellularor organ compartment, tissue, organ or region of the body, as, e.g.,compared to a species absent such a ligand. Preferred ligands will nottake part in duplex pairing in a duplexed nucleic acid.

Ligands can include a naturally occurring substance, such as a protein(e.g., human serum albumin (HSA), low-density lipoprotein (LDL), orglobulin); carbohydrate (e.g., a dextran, pullulan, chitin, chitosan,inulin, cyclodextrin or hyaluronic acid); or a lipid. The ligand canalso be a recombinant or synthetic molecule, such as a syntheticpolymer, e.g., a synthetic polyamino acid. Examples of polyamino acidsinclude polyamino acid is a polylysine (PLL), poly L-aspartic acid, polyL-glutamic acid, styrene-maleic acid anhydride copolymer,poly(L-lactide-co-glycolied) copolymer, divinyl ether-maleic anhydridecopolymer, N-(2-hydroxypropyl)methacrylamide copolymer (HMPA),polyethylene glycol (PEG), polyvinyl alcohol (PVA), polyurethane,poly(2-ethylacryllic acid), N-isopropylacrylamide polymers, orpolyphosphazine. Example of polyamines include: polyethylenimine,polylysine (PLL), spermine, spermidine, polyamine,pseudopeptide-polyamine, peptidomimetic polyamine, dendrimer polyamine,arginine, amidine, protamine, cationic lipid, cationic porphyrin,quaternary salt of a polyamine, or an alpha helical peptide.

Ligands can also include targeting groups, e.g., a cell or tissuetargeting agent, e.g., a lectin, glycoprotein, lipid or protein, e.g.,an antibody, that binds to a specified cell type such as a kidney cell.A targeting group can be a thyrotropin, melanotropin, lectin,glycoprotein, surfactant protein A, Mucin carbohydrate, multivalentlactose, multivalent galactose, N-acetyl-galactosamine,N-acetyl-gulucosamine multivalent mannose, multivalent fucose,glycosylated polyaminoacids, multivalent galactose, transferrin,bisphosphonate, polyglutamate, polyaspartate, a lipid, cholesterol, asteroid, bile acid, folate, vitamin B12, vitamin A, biotin, or an RGDpeptide or RGD peptide mimetic.

Other examples of ligands include dyes, intercalating agents (e.g.acridines), cross-linkers (e.g. psoralene, mitomycin C), porphyrins(TPPC4, texaphyrin, Sapphyrin), polycyclic aromatic hydrocarbons (e.g.,phenazine, dihydrophenazine), artificial endonucleases (e.g. EDTA),lipophilic molecules, e.g., cholesterol, cholic acid, adamantane aceticacid, 1-pyrene butyric acid, dihydrotestosterone,1,3-Bis-O(hexadecyl)glycerol, geranyloxyhexyl group, hexadecylglycerol,borneol, menthol, 1,3-propanediol, heptadecyl group, palmitic acid,myristic acid, O3-(oleoyl)lithocholic acid, O3-(oleoyl)cholenic acid,dimethoxytrityl, or phenoxazine) and peptide conjugates (e.g.,antennapedia peptide, Tat peptide), alkylating agents, phosphate, amino,mercapto, PEG (e.g., PEG-40K), MPEG, [MPEG]2, polyamino, alkyl,substituted alkyl, radiolabeled markers, enzymes, haptens (e.g. biotin),transport/absorption facilitators (e.g., aspirin, vitamin E, folicacid), synthetic ribonucleases (e.g., imidazole, bisimidazole,histamine, imidazole clusters, acridine-imidazole conjugates, Eu3+complexes of tetraazamacrocycles), dinitrophenyl, HRP, or AP.

Ligands can be proteins, e.g., glycoproteins, or peptides, e.g.,molecules having a specific affinity for a co-ligand, or antibodiese.g., an antibody, that binds to a specified cell type such as a hepaticcell. Ligands can also include hormones and hormone receptors. They canalso include non-peptidic species, such as lipids, lectins,carbohydrates, vitamins, cofactors, multivalent lactose, multivalentgalactose, N-acetyl-galactosamine, N-acetyl-gulucosamine multivalentmannose, or multivalent fucose. The ligand can be, for example, alipopolysaccharide, an activator of p38 MAP kinase, or an activator ofNF-κB.

The ligand can be a substance, e.g., a drug, which can increase theuptake of the iRNA agent into the cell, for example, by disrupting thecell's cytoskeleton, e.g., by disrupting the cell's microtubules,microfilaments, and/or intermediate filaments. The drug can be, forexample, taxon, vincristine, vinblastine, cytochalasin, nocodazole,japlakinolide, latrunculin A, phalloidin, swinholide A, indanocine, ormyoservin.

In some embodiments, a ligand attached to an iRNA as described hereinacts as a pharmacokinetic (PK) modulator. As used herein, a “PKmodulator” refers to a pharmacokinetic modulator. PK modulators includelipophiles, bile acids, steroids, phospholipid analogues, peptides,protein binding agents, PEG, vitamins etc. Exemplary PK modulatorsinclude, but are not limited to, cholesterol, fatty acids, cholic acid,lithocholic acid, dialkylglycerides, diacylglyceride, phospholipids,sphingolipids, naproxen, ibuprofen, vitamin E, biotin etc.Oligonucleotides that comprise a number of phosphorothioate linkages arealso known to bind to serum protein, thus short oligonucleotides, e.g.,oligonucleotides of about 5 bases, 10 bases, 15 bases or 20 bases,comprising multiple of phosphorothioate linkages in the backbone arealso amenable to the technology described herein as ligands (e.g. as PKmodulating ligands). In addition, aptamers that bind serum components(e.g. serum proteins) are also suitable for use as PK modulating ligandsin the embodiments described herein.

Lipid Conjugates

In one embodiment, the ligand or conjugate is a lipid or lipid-basedmolecule. Such a lipid or lipid-based molecule preferably binds a serumprotein, e.g., human serum albumin (HSA). An HSA binding ligand allowsfor distribution of the conjugate to a target tissue, e.g., a non-kidneytarget tissue of the body. For example, the target tissue can be theliver, including parenchymal cells of the liver. Other molecules thatcan bind HSA can also be used as ligands. For example, neproxin oraspirin can be used. A lipid or lipid-based ligand can (a) increaseresistance to degradation of the conjugate, (b) increase targeting ortransport into a target cell or cell membrane, and/or (c) can be used toadjust binding to a serum protein, e.g., HSA.

A lipid based ligand can be used to inhibit, e.g., control the bindingof the conjugate to a target tissue. For example, a lipid or lipid-basedligand that binds to HSA more strongly will be less likely to betargeted to the kidney and therefore less likely to be cleared from thebody. A lipid or lipid-based ligand that binds to HSA less strongly canbe used to target the conjugate to the kidney.

In a preferred embodiment, the lipid based ligand binds HSA. Preferably,it binds HSA with a sufficient affinity such that the conjugate will bepreferably distributed to a non-kidney tissue. However, it is preferredthat the affinity not be so strong that the HSA-ligand binding cannot bereversed.

In another preferred embodiment, the lipid based ligand binds HSA weaklyor not at all, such that the conjugate will be preferably distributed tothe kidney. Other moieties that target to kidney cells can also be usedin place of or in addition to the lipid based ligand.

In another aspect, the ligand is a moiety, e.g., a vitamin, which istaken up by a target cell, e.g., a proliferating cell. These areparticularly useful for treating disorders characterized by unwantedcell proliferation, e.g., of the malignant or non-malignant type, e.g.,cancer cells. Exemplary vitamins include vitamin A, E, and K. Otherexemplary vitamins include are B vitamin, e.g., folic acid, B12,riboflavin, biotin, pyridoxal or other vitamins or nutrients taken up bytarget cells such as liver cells. Also included are HSA and low densitylipoprotein (LDL).

Cell Permeation Peptide and Agents

In another aspect, the ligand is a cell-permeation agent, preferably ahelical cell-permeation agent. Preferably, the agent is amphipathic. Anexemplary agent is a peptide such as tat or antennopedia. If the agentis a peptide, it can be modified, including a peptidylmimetic,invertomers, non-peptide or pseudo-peptide linkages, and use of D-aminoacids. The helical agent is preferably an alpha-helical agent, whichpreferably has a lipophilic and a lipophobic phase.

The ligand can be a peptide or peptidomimetic. A peptidomimetic (alsoreferred to herein as an oligopeptidomimetic) is a molecule capable offolding into a defined three-dimensional structure similar to a naturalpeptide. The attachment of peptide and peptidomimetics to iRNA agentscan affect pharmacokinetic distribution of the iRNA, such as byenhancing cellular recognition and absorption. The peptide orpeptidomimetic moiety can be about 5-50 amino acids long, e.g., about 5,10, 15, 20, 25, 30, 35, 40, 45, or 50 amino acids long.

A peptide or peptidomimetic can be, for example, a cell permeationpeptide, cationic peptide, amphipathic peptide, or hydrophobic peptide(e.g., consisting primarily of Tyr, Trp or Phe). The peptide moiety canbe a dendrimer peptide, constrained peptide or crosslinked peptide. Inanother alternative, the peptide moiety can include a hydrophobicmembrane translocation sequence (MTS). An exemplary hydrophobicMTS-containing peptide is RFGF having the amino acid sequenceAAVALLPAVLLALLAP (SEQ ID NO: 25). An RFGF analogue (e.g., amino acidsequence AALLPVLLAAP (SEQ ID NO: 26) containing a hydrophobic MTS canalso be a targeting moiety. The peptide moiety can be a “delivery”peptide, which can carry large polar molecules including peptides,oligonucleotides, and protein across cell membranes. For example,sequences from the HIV Tat protein (GRKKRRQRRRPPQ (SEQ ID NO: 27) andthe Drosophila Antennapedia protein (RQIKIWFQNRRMKWKK (SEQ ID NO: 28)have been found to be capable of functioning as delivery peptides. Apeptide or peptidomimetic can be encoded by a random sequence of DNA,such as a peptide identified from a phage-display library, orone-bead-one-compound (OBOC) combinatorial library (Lam et al., Nature,354:82-84, 1991). Examples of a peptide or peptidomimetic tethered to adsRNA agent via an incorporated monomer unit for cell targeting purposesis an arginine-glycine-aspartic acid (RGD)-peptide, or RGD mimic. Apeptide moiety can range in length from about 5 amino acids to about 40amino acids. The peptide moieties can have a structural modification,such as to increase stability or direct conformational properties. Anyof the structural modifications described below can be utilized.

An RGD peptide moiety can be used to target a tumor cell, such as anendothelial tumor cell or a breast cancer tumor cell (Zitzmann et al.,Cancer Res., 62:5139-43, 2002). An RGD peptide can facilitate targetingof a dsRNA agent to tumors of a variety of other tissues, including thelung, kidney, spleen, or liver (Aoki et al., Cancer Gene Therapy8:783-787, 2001). Preferably, the RGD peptide will facilitate targetingof an iRNA agent to the kidney. The RGD peptide can be linear or cyclic,and can be modified, e.g., glycosylated or methylated to facilitatetargeting to specific tissues. For example, a glycosylated RGD peptidecan deliver an iRNA agent to a tumor cell expressing αvβ₃ (Haubner etal., Jour. Nucl. Med., 42:326-336, 2001).

A “cell permeation peptide” is capable of permeating a cell, e.g., amicrobial cell, such as a bacterial or fungal cell, or a mammalian cell,such as a human cell. A microbial cell-permeating peptide can be, forexample, a α-helical linear peptide (e.g., LL-37 or Ceropin P1), adisulfide bond-containing peptide (e.g., α-defensin, β-defensin orbactenecin), or a peptide containing only one or two dominating aminoacids (e.g., PR-39 or indolicidin). A cell permeation peptide can alsoinclude a nuclear localization signal (NLS). For example, a cellpermeation peptide can be a bipartite amphipathic peptide, such as MPG,which is derived from the fusion peptide domain of HIV-1 gp41 and theNLS of SV40 large T antigen (Simeoni et al., Nucl. Acids Res.31:2717-2724, 2003).

Carbohydrate Conjugates

In some embodiments, the iRNA oligonucleotides described herein furthercomprise carbohydrate conjugates. The carbohydrate conjugates areadvantageous for the in vivo delivery of nucleic acids, as well ascompositions suitable for in vivo therapeutic use, as described herein.As used herein, “carbohydrate” refers to a compound which is either acarbohydrate per se made up of one or more monosaccharide units havingat least 6 carbon atoms (which can be linear, branched or cyclic) withan oxygen, nitrogen or sulfur atom bonded to each carbon atom; or acompound having as a part thereof a carbohydrate moiety made up of oneor more monosaccharide units each having at least six carbon atoms(which can be linear, branched or cyclic), with an oxygen, nitrogen orsulfur atom bonded to each carbon atom. Representative carbohydratesinclude the sugars (mono-, di-, tri- and oligosaccharides containingfrom about 4-9 monosaccharide units), and polysaccharides such asstarches, glycogen, cellulose and polysaccharide gums. Specificmonosaccharides include C5 and above (preferably C5-C8) sugars; di- andtrisaccharides include sugars having two or three monosaccharide units(preferably C5-C8).

In one embodiment, the carbohydrate conjugate is selected from the groupconsisting of:

i.e., Formula II—Formula XXII.

Another representative carbohydrate conjugate for use in the embodimentsdescribed herein includes, but is not limited to,

when one of X or Y is an oligonucleotide, the other is a hydrogen.

In some embodiments, the carbohydrate conjugate further comprises otherligand such as, but not limited to, PK modulator, endosomolytic ligand,and cell permeation peptide.

Linkers

In some embodiments, the conjugates described herein can be attached tothe iRNA oligonucleotide with various linkers that can be cleavable ornon cleavable.

The term “linker” or “linking group” means an organic moiety thatconnects two parts of a compound. Linkers typically comprise a directbond or an atom such as oxygen or sulfur, a unit such as NR8, C(O),C(O)NH, SO, SO2, SO2NH or a chain of atoms, such as, but not limited to,substituted or unsubstituted alkyl, substituted or unsubstitutedalkenyl, substituted or unsubstituted alkynyl, arylalkyl, arylalkenyl,arylalkynyl, heteroarylalkyl, heteroarylalkenyl, heteroarylalkynyl,heterocyclylalkyl, heterocyclylalkenyl, heterocyclylalkynyl, aryl,heteroaryl, heterocyclyl, cycloalkyl, cycloalkenyl, alkylarylalkyl,alkylarylalkenyl, alkylarylalkynyl, alkenylarylalkyl,alkenylarylalkenyl, alkenylarylalkynyl, alkynylarylalkyl,alkynylarylalkenyl, alkynylarylalkynyl, alkylheteroarylalkyl,alkylheteroarylalkenyl, alkylheteroarylalkynyl, alkenylheteroarylalkyl,alkenylheteroarylalkenyl, alkenylheteroarylalkynyl,alkynylheteroarylalkyl, alkynylheteroarylalkenyl,alkynylheteroarylalkynyl, alkylheterocyclylalkyl,alkylheterocyclylalkenyl, alkylhererocyclylalkynyl,alkenylheterocyclylalkyl, alkenylheterocyclylalkenyl,alkenylheterocyclylalkynyl, alkynylheterocyclylalkyl,alkynylheterocyclylalkenyl, alkynylheterocyclylalkynyl, alkylaryl,alkenylaryl, alkynylaryl, alkylheteroaryl, alkenylheteroaryl,alkynylhereroaryl, which one or more methylenes can be interrupted orterminated by O, S, S(O), SO2, N(R8), C(O), substituted or unsubstitutedaryl, substituted or unsubstituted heteroaryl, substituted orunsubstituted heterocyclic; where R8 is hydrogen, acyl, aliphatic orsubstituted aliphatic. In one embodiment, the linker is between 1-24atoms, preferably 4-24 atoms, preferably 6-18 atoms, more preferably8-18 atoms, and most preferably 8-16 atoms.

A cleavable linking group is one which is sufficiently stable outsidethe cell, but which upon entry into a target cell is cleaved to releasethe two parts the linker is holding together. In a preferred embodiment,the cleavable linking group is cleaved at least 10 times or more,preferably at least 100 times faster in the target cell or under a firstreference condition (which can, e.g., be selected to mimic or representintracellular conditions) than in the blood of a subject, or under asecond reference condition (which can, e.g., be selected to mimic orrepresent conditions found in the blood or serum).

Cleavable linking groups are susceptible to cleavage agents, e.g., pH,redox potential or the presence of degradative molecules. Generally,cleavage agents are more prevalent or found at higher levels oractivities inside cells than in serum or blood. Examples of suchdegradative agents include: redox agents which are selected forparticular substrates or which have no substrate specificity, including,e.g., oxidative or reductive enzymes or reductive agents such asmercaptans, present in cells, that can degrade a redox cleavable linkinggroup by reduction; esterases; endosomes or agents that can create anacidic environment, e.g., those that result in a pH of five or lower;enzymes that can hydrolyze or degrade an acid cleavable linking group byacting as a general acid, peptidases (which can be substrate specific),and phosphatases.

A cleavable linkage group, such as a disulfide bond can be susceptibleto pH. The pH of human serum is 7.4, while the average intracellular pHis slightly lower, ranging from about 7.1-7.3. Endosomes have a moreacidic pH, in the range of 5.5-6.0, and lysosomes have an even moreacidic pH at around 5.0. Some linkers will have a cleavable linkinggroup that is cleaved at a preferred pH, thereby releasing the cationiclipid from the ligand inside the cell, or into the desired compartmentof the cell.

A linker can include a cleavable linking group that is cleavable by aparticular enzyme. The type of cleavable linking group incorporated intoa linker can depend on the cell to be targeted. For example, livertargeting ligands can be linked to the cationic lipids through a linkerthat includes an ester group. Liver cells are rich in esterases, andtherefore the linker will be cleaved more efficiently in liver cellsthan in cell types that are not esterase-rich. Other cell-types rich inesterases include cells of the lung, renal cortex, and testis.

Linkers that contain peptide bonds can be used when targeting cell typesrich in peptidases, such as liver cells and synoviocytes.

In general, the suitability of a candidate cleavable linking group canbe evaluated by testing the ability of a degradative agent (orcondition) to cleave the candidate linking group. It will also bedesirable to also test the candidate cleavable linking group for theability to resist cleavage in the blood or when in contact with othernon-target tissue. Thus one can determine the relative susceptibility tocleavage between a first and a second condition, where the first isselected to be indicative of cleavage in a target cell and the second isselected to be indicative of cleavage in other tissues or biologicalfluids, e.g., blood or serum. The evaluations can be carried out in cellfree systems, in cells, in cell culture, in organ or tissue culture, orin whole animals. It can be useful to make initial evaluations incell-free or culture conditions and to confirm by further evaluations inwhole animals. In preferred embodiments, useful candidate compounds arecleaved at least 2, 4, 10 or 100 times faster in the cell (or under invitro conditions selected to mimic intracellular conditions) as comparedto blood or serum (or under in vitro conditions selected to mimicextracellular conditions).

Redox Cleavable Linking Groups

One class of cleavable linking groups are redox cleavable linking groupsthat are cleaved upon reduction or oxidation. An example of reductivelycleavable linking group is a disulphide linking group (—S—S—). Todetermine if a candidate cleavable linking group is a suitable“reductively cleavable linking group,” or for example is suitable foruse with a particular iRNA moiety and particular targeting agent one canlook to methods described herein. For example, a candidate can beevaluated by incubation with dithiothreitol (DTT), or other reducingagent using reagents know in the art, which mimic the rate of cleavagewhich would be observed in a cell, e.g., a target cell. The candidatescan also be evaluated under conditions which are selected to mimic bloodor serum conditions. In a preferred embodiment, candidate compounds arecleaved by at most 10% in the blood. In preferred embodiments, usefulcandidate compounds are degraded at least 2, 4, 10 or 100 times fasterin the cell (or under in vitro conditions selected to mimicintracellular conditions) as compared to blood (or under in vitroconditions selected to mimic extracellular conditions). The rate ofcleavage of candidate compounds can be determined using standard enzymekinetics assays under conditions chosen to mimic intracellular media andcompared to conditions chosen to mimic extracellular media.

Phosphate-Based Cleavable Linking Groups

Phosphate-based cleavable linking groups are cleaved by agents thatdegrade or hydrolyze the phosphate group. An example of an agent thatcleaves phosphate groups in cells are enzymes such as phosphatases incells. Examples of phosphate-based linking groups are —O—P(O)(ORk)-O—,—O—P(S)(ORk)-O—, —O—P(S)(SRk)-O—, —S—P(O)(ORk)-O—, —O—P(O)(ORk)-S—,—S—P(O)(ORk)-S—, —O—P(S)(ORk)-S—, —S—P(S)(ORk)-O—, —O—P(O)(Rk)-O—,—O—P(S)(Rk)-O—, —S—P(O)(Rk)-O—, —S—P(S)(Rk)-O—, —S—P(O)(Rk)-S—,—O—P(S)(Rk)-S—. Preferred embodiments are —O—P(O)(OH)—O—,—O—P(S)(OH)—O—, —O—P(S)(SH)—O—, —S—P(O)(OH)—O—, —O—P(O)(OH)—S—,—S—P(O)(OH)—S—, —O—P(S)(OH)—S—, —S—P(S)(OH)—O—, —O—P(O)(H)—O—,—O—P(S)(H)—O—, —S—P(O)(H)—O—, —S—P(S)(H)—O—, —S—P(O)(H)—S—,—O—P(S)(H)—S—. A preferred embodiment is —O—P(O)(OH)—O—. Thesecandidates can be evaluated using methods analogous to those describedabove.

Acid Cleavable Linking Groups

Acid cleavable linking groups are linking groups that are cleaved underacidic conditions. In preferred embodiments acid cleavable linkinggroups are cleaved in an acidic environment with a pH of about 6.5 orlower (e.g., about 6.0, 5.5, 5.0, or lower), or by agents such asenzymes that can act as a general acid. In a cell, specific low pHorganelles, such as endosomes and lysosomes can provide a cleavingenvironment for acid cleavable linking groups. Examples of acidcleavable linking groups include but are not limited to hydrazones,esters, and esters of amino acids. Acid cleavable groups can have thegeneral formula —C═NN—, C(O)O, or —OC(O). A preferred embodiment is whenthe carbon attached to the oxygen of the ester (the alkoxy group) is anaryl group, substituted alkyl group, or tertiary alkyl group such asdimethyl pentyl or t-butyl. These candidates can be evaluated usingmethods analogous to those described above.

Ester-Based Linking Groups

Ester-based cleavable linking groups are cleaved by enzymes such asesterases and amidases in cells. Examples of ester-based cleavablelinking groups include but are not limited to esters of alkylene,alkenylene and alkynylene groups. Ester cleavable linking groups havethe general formula —C(O)O—, or —OC(O)—. These candidates can beevaluated using methods analogous to those described above.

Peptide-Based Cleaving Groups

Peptide-based cleavable linking groups are cleaved by enzymes such aspeptidases and proteases in cells. Peptide-based cleavable linkinggroups are peptide bonds formed between amino acids to yieldoligopeptides (e.g., dipeptides, tripeptides etc.) and polypeptides.Peptide-based cleavable groups do not include the amide group(—C(O)NH—). The amide group can be formed between any alkylene,alkenylene or alkynelene. A peptide bond is a special type of amide bondformed between amino acids to yield peptides and proteins. The peptidebased cleavage group is generally limited to the peptide bond (i.e., theamide bond) formed between amino acids yielding peptides and proteinsand does not include the entire amide functional group. Peptide-basedcleavable linking groups have the general formula—NHCHRAC(O)NHCHRBC(O)—, where RA and RB are the R groups of the twoadjacent amino acids. These candidates can be evaluated using methodsanalogous to those described above.

Representative carbohydrate conjugates with linkers include, but are notlimited to,

when one of X or Y is an oligonucleotide, the other is a hydrogen.

Representative U.S. patents that teach the preparation of RNA conjugatesinclude, but are not limited to, U.S. Pat. Nos. 4,828,979; 4,948,882;5,218,105; 5,525,465; 5,541,313; 5,545,730; 5,552,538; 5,578,717,5,580,731; 5,591,584; 5,109,124; 5,118,802; 5,138,045; 5,414,077;5,486,603; 5,512,439; 5,578,718; 5,608,046; 4,587,044; 4,605,735;4,667,025; 4,762,779; 4,789,737; 4,824,941; 4,835,263; 4,876,335;4,904,582; 4,958,013; 5,082,830; 5,112,963; 5,214,136; 5,082,830;5,112,963; 5,214,136; 5,245,022; 5,254,469; 5,258,506; 5,262,536;5,272,250; 5,292,873; 5,317,098; 5,371,241, 5,391,723; 5,416,203,5,451,463; 5,510,475; 5,512,667; 5,514,785; 5,565,552; 5,567,810;5,574,142; 5,585,481; 5,587,371; 5,595,726; 5,597,696; 5,599,923;5,599,928 and 5,688,941; 6,294,664; 6,320,017; 6,576,752; 6,783,931;6,900,297; 7,037,646; each of which is herein incorporated by reference.

It is not necessary for all positions in a given compound to beuniformly modified, and in fact more than one of the aforementionedmodifications can be incorporated in a single compound or even at asingle nucleoside within an iRNA. The technology described herein alsoincludes iRNA compounds that are chimeric compounds. “Chimeric” iRNAcompounds or “chimeras,” in the context of this invention, are iRNAcompounds, preferably dsRNAs, which contain two or more chemicallydistinct regions, each made up of at least one monomer unit, i.e., anucleotide in the case of a dsRNA compound. These iRNAs typicallycontain at least one region wherein the RNA is modified so as to conferupon the iRNA increased resistance to nuclease degradation, increasedcellular uptake, and/or increased binding affinity for the targetnucleic acid. An additional region of the iRNA can serve as a substratefor enzymes capable of cleaving RNA:DNA or RNA:RNA hybrids. By way ofexample, RNase H is a cellular endonuclease which cleaves the RNA strandof an RNA:DNA duplex. Activation of RNase H, therefore, results incleavage of the RNA target, thereby greatly enhancing the efficiency ofiRNA inhibition of gene expression. Consequently, comparable results canoften be obtained with shorter iRNAs when chimeric dsRNAs are used,compared to phosphorothioate deoxy dsRNAs hybridizing to the same targetregion. Cleavage of the RNA target can be routinely detected by gelelectrophoresis and, if necessary, associated nucleic acid hybridizationtechniques known in the art.

In certain instances, the RNA of an iRNA can be modified by a non-ligandgroup. A number of non-ligand molecules have been conjugated to iRNAs inorder to enhance the activity, cellular distribution or cellular uptakeof the iRNA, and procedures for performing such conjugations areavailable in the scientific literature. Such non-ligand moieties haveincluded lipid moieties, such as cholesterol (Kubo, T. et al., Biochem.Biophys. Res. Comm., 2007, 365(1):54-61; Letsinger et al., Proc. Natl.Acad. Sci. USA, 1989, 86:6553), cholic acid (Manoharan et al., Bioorg.Med. Chem. Lett., 1994, 4:1053), a thioether, e.g., hexyl-S-tritylthiol(Manoharan et al., Ann. N.Y. Acad. Sci., 1992, 660:306; Manoharan etal., Bioorg. Med. Chem. Let., 1993, 3:2765), a thiocholesterol(Oberhauser et al., Nucl. Acids Res., 1992, 20:533), an aliphatic chain,e.g., dodecandiol or undecyl residues (Saison-Behmoaras et al., EMBO J.,1991, 10:111; Kabanov et al., FEBS Lett., 1990, 259:327; Svinarchuk etal., Biochimie, 1993, 75:49), a phospholipid, e.g.,di-hexadecyl-rac-glycerol or triethylammonium1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate (Manoharan et al.,Tetrahedron Lett., 1995, 36:3651; Shea et al., Nucl. Acids Res., 1990,18:3777), a polyamine or a polyethylene glycol chain (Manoharan et al.,Nucleosides & Nucleotides, 1995, 14:969), or adamantane acetic acid(Manoharan et al., Tetrahedron Lett., 1995, 36:3651), a palmityl moiety(Mishra et al., Biochim. Biophys. Acta, 1995, 1264:229), or anoctadecylamine or hexylamino-carbonyl-oxycholesterol moiety (Crooke etal., J. Pharmacol. Exp. Ther., 1996, 277:923). Representative UnitedStates patents that teach the preparation of such RNA conjugates havebeen listed above. Typical conjugation protocols involve the synthesisof an RNAs bearing an aminolinker at one or more positions of thesequence. The amino group is then reacted with the molecule beingconjugated using appropriate coupling or activating reagents. Theconjugation reaction can be performed either with the RNA still bound tothe solid support or following cleavage of the RNA, in solution phase.Purification of the RNA conjugate by HPLC typically affords the pureconjugate.

Delivery of iRNA

The delivery of an iRNA to a subject in need thereof can be achieved ina number of different ways. In vivo delivery can be performed directlyby administering a composition comprising an iRNA, e.g. a dsRNA, to asubject. Alternatively, delivery can be performed indirectly byadministering one or more vectors that encode and direct the expressionof the iRNA. These alternatives are discussed further below.

Delivery of an iRNA Composition

In general, any method of delivering a nucleic acid molecule can beadapted for use with an iRNA (see e.g., Akhtar S. and Julian R L. (1992)Trends Cell. Biol. 2(5):139-144 and WO94/02595, which are incorporatedherein by reference in their entireties). However, there are threefactors that are important to consider in order to successfully deliveran iRNA molecule in vivo: (a) biological stability of the deliveredmolecule, (2) preventing non-specific effects, and (3) accumulation ofthe delivered molecule in the target tissue. The non-specific effects ofan iRNA can be minimized by local administration, for example by directinjection or implantation into a tissue (as a non-limiting example, atumor) or topically administering the preparation. Local administrationto a treatment site maximizes local concentration of the agent, limitsthe exposure of the agent to systemic tissues that can otherwise beharmed by the agent or that can degrade the agent, and permits a lowertotal dose of the iRNA molecule to be administered. Several studies haveshown successful knockdown of gene products when an iRNA is administeredlocally. For example, intraocular delivery of a VEGF dsRNA byintravitreal injection in cynomolgus monkeys (Tolentino, M J., et al(2004) Retina 24:132-138) and subretinal injections in mice (Reich, SJ., et al (2003) Mol. Vis. 9:210-216) were both shown to preventneovascularization in an experimental model of age-related maculardegeneration. In addition, direct intratumoral injection of a dsRNA inmice reduces tumor volume (Pille, J., et al (2005) Mol. Ther.11:267-274) and can prolong survival of tumor-bearing mice (Kim, W J.,et al (2006) Mol. Ther. 14:343-350; Li, S., et al (2007) Mol. Ther.15:515-523). RNA interference has also shown success with local deliveryto the CNS by direct injection (Dorn, G., et al. (2004) Nucleic Acids32:e49; Tan, P H., et al (2005) Gene Ther. 12:59-66; Makimura, H., et al(2002) BMC Neurosci. 3:18; Shishkina, G T., et al (2004) Neuroscience129:521-528; Thakker, E R., et al (2004) Proc. Natl. Acad. Sci. U.S.A.101:17270-17275; Akaneya, Y., et al (2005) J. Neurophysiol. 93:594-602)and to the lungs by intranasal administration (Howard, K A., et al(2006) Mol. Ther. 14:476-484; Zhang, X., et al (2004) J. Biol. Chem.279:10677-10684; Bitko, V., et al (2005) Nat. Med. 11:50-55). Foradministering an iRNA systemically for the treatment of a disease, theRNA can be modified or alternatively delivered using a drug deliverysystem; both methods act to prevent the rapid degradation of the dsRNAby endo- and exo-nucleases in vivo. Modification of the RNA or thepharmaceutical carrier can also permit targeting of the iRNA compositionto the target tissue and avoid undesirable off-target effects. iRNAmolecules can be modified by chemical conjugation to lipophilic groupssuch as cholesterol to enhance cellular uptake and prevent degradation.For example, an iRNA directed against ApoB conjugated to a lipophiliccholesterol moiety was injected systemically into mice and resulted inknockdown of apoB mRNA in both the liver and jejunum (Soutschek, J., etal (2004) Nature 432:173-178). Conjugation of an iRNA to an aptamer hasbeen shown to inhibit tumor growth and mediate tumor regression in amouse model of prostate cancer (McNamara, J O., et al (2006) Nat.Biotechnol. 24:1005-1015). In an alternative embodiment, the iRNA can bedelivered using drug delivery systems such as a nanoparticle, adendrimer, a polymer, liposomes, or a cationic delivery system.Positively charged cationic delivery systems facilitate binding of aniRNA molecule (negatively charged) and also enhance interactions at thenegatively charged cell membrane to permit efficient uptake of an iRNAby the cell. Cationic lipids, dendrimers, or polymers can either bebound to an iRNA, or induced to form a vesicle or micelle (see e.g., KimS H., et al (2008) Journal of Controlled Release 129(2):107-116) thatencases an iRNA. The formation of vesicles or micelles further preventsdegradation of the iRNA when administered systemically. Methods formaking and administering cationic-iRNA complexes are well within theabilities of one skilled in the art (see e.g., Sorensen, D R., et al(2003) J. Mol. Biol 327:761-766; Verma, U N., et al (2003) Clin. CancerRes. 9:1291-1300; Arnold, A S et al (2007) J. Hypertens. 25:197-205,which are incorporated herein by reference in their entirety). Somenon-limiting examples of drug delivery systems useful for systemicdelivery of iRNAs include DOTAP (Sorensen, D R., et al (2003), supra;Verma, U N., et al (2003), supra), Oligofectamine, “solid nucleic acidlipid particles” (Zimmermann, T S., et al (2006) Nature 441:111-114),cardiolipin (Chien, P Y., et al (2005) Cancer Gene Ther. 12:321-328;Pal, A., et al (2005) Int J. Oncol. 26:1087-1091), polyethyleneimine(Bonnet M E., et al (2008) Pharm. Res. August 16 Epub ahead of print;Aigner, A. (2006) J. Biomed. Biotechnol. 71659), Arg-Gly-Asp (RGD)peptides (Liu, S. (2006) Mol. Pharm. 3:472-487), and polyamidoamines(Tomalia, D A., et al (2007) Biochem. Soc. Trans. 35:61-67; Yoo, H., etal (1999) Pharm. Res. 16:1799-1804). In some embodiments, an iRNA formsa complex with cyclodextrin for systemic administration. Methods foradministration and pharmaceutical compositions of iRNAs andcyclodextrins can be found in U.S. Pat. No. 7,427,605, which is hereinincorporated by reference in its entirety.

Vector Encoded dsRNAs

In another aspect, iRNA targeting the Serpina1 gene can be expressedfrom transcription units inserted into DNA or RNA vectors (see, e.g.,Couture, A, et al., TIG. (1996), 12:5-10; Skillern, A., et al.,International PCT Publication No. WO 00/22113, Conrad, International PCTPublication No. WO 00/22114, and Conrad, U.S. Pat. No. 6,054,299).Expression can be transient (on the order of hours to weeks) orsustained (weeks to months or longer), depending upon the specificconstruct used and the target tissue or cell type. These transgenes canbe introduced as a linear construct, a circular plasmid, or a viralvector, which can be an integrating or non-integrating vector. Thetransgene can also be constructed to permit it to be inherited as anextrachromosomal plasmid (Gassmann, et al., Proc. Natl. Acad. Sci. USA(1995) 92:1292).

The individual strand or strands of an iRNA can be transcribed from apromoter on an expression vector. Where two separate strands are to beexpressed to generate, for example, a dsRNA, two separate expressionvectors can be co-introduced (e.g., by transfection or infection) into atarget cell. Alternatively each individual strand of a dsRNA can betranscribed by promoters both of which are located on the sameexpression plasmid. In one embodiment, a dsRNA is expressed as invertedrepeat polynucleotides joined by a linker polynucleotide sequence suchthat the dsRNA has a stem and loop structure.

iRNA expression vectors are generally DNA plasmids or viral vectors.Expression vectors compatible with eukaryotic cells, preferably thosecompatible with vertebrate cells, can be used to produce recombinantconstructs for the expression of an iRNA as described herein. Eukaryoticcell expression vectors are well known in the art and are available froma number of commercial sources. Typically, such vectors are providedcontaining convenient restriction sites for insertion of the desirednucleic acid segment. Delivery of iRNA expressing vectors can besystemic, such as by intravenous or intramuscular administration, byadministration to target cells ex-planted from the patient followed byreintroduction into the patient, or by any other means that allows forintroduction into a desired target cell.

iRNA expression plasmids can be transfected into target cells as acomplex with cationic lipid carriers (e.g., Oligofectamine) ornon-cationic lipid-based carriers (e.g., Transit-TKO™). Multiple lipidtransfections for iRNA-mediated knockdowns targeting different regionsof a target RNA over a period of a week or more are also contemplated.Successful introduction of vectors into host cells can be monitoredusing various known methods. For example, transient transfection can besignaled with a reporter, such as a fluorescent marker, such as GreenFluorescent Protein (GFP). Stable transfection of cells ex vivo can beensured using markers that provide the transfected cell with resistanceto specific environmental factors (e.g., antibiotics and drugs), such ashygromycin B resistance.

Viral vector systems which can be utilized with the methods andcompositions described herein include, but are not limited to, (a)adenovirus vectors; (b) retrovirus vectors, including but not limited tolentiviral vectors, moloney murine leukemia virus, etc.; (c)adeno-associated virus vectors; (d) herpes simplex virus vectors; (e) SV40 vectors; (f) polyoma virus vectors; (g) papilloma virus vectors; (h)picornavirus vectors; (i) pox virus vectors such as an orthopox, e.g.,vaccinia virus vectors or avipox, e.g. canary pox or fowl pox; and (j) ahelper-dependent or gutless adenovirus. Replication-defective virusescan also be advantageous. Different vectors will or will not becomeincorporated into the cells' genome. The constructs can include viralsequences for transfection, if desired. Alternatively, the construct canbe incorporated into vectors capable of episomal replication, e.g. EPVand EBV vectors. Constructs for the recombinant expression of an iRNAwill generally require regulatory elements, e.g., promoters, enhancers,etc., to ensure the expression of the iRNA in target cells. Otheraspects to consider for vectors and constructs are further describedbelow.

Vectors useful for the delivery of an iRNA will include regulatoryelements (promoter, enhancer, etc.) sufficient for expression of theiRNA in the desired target cell or tissue. The regulatory elements canbe chosen to provide either constitutive or regulated/inducibleexpression.

Expression of the iRNA can be precisely regulated, for example, by usingan inducible regulatory sequence that is sensitive to certainphysiological regulators, e.g., circulating glucose levels, or hormones(Docherty et al., 1994, FASEB J. 8:20-24). Such inducible expressionsystems, suitable for the control of dsRNA expression in cells or inmammals include, for example, regulation by ecdysone, by estrogen,progesterone, tetracycline, chemical inducers of dimerization, andisopropyl-beta-D1-thiogalactopyranoside (IPTG). A person skilled in theart would be able to choose the appropriate regulatory/promoter sequencebased on the intended use of the iRNA transgene.

In a specific embodiment, viral vectors that contain nucleic acidsequences encoding an iRNA can be used. For example, a retroviral vectorcan be used (see Miller et al., Meth. Enzymol. 217:581-599 (1993)).These retroviral vectors contain the components necessary for thecorrect packaging of the viral genome and integration into the host cellDNA. The nucleic acid sequences encoding an iRNA are cloned into one ormore vectors, which facilitates delivery of the nucleic acid into apatient. More detail about retroviral vectors can be found, for example,in Boesen et al., Biotherapy 6:291-302 (1994), which describes the useof a retroviral vector to deliver the mdrl gene to hematopoietic stemcells in order to make the stem cells more resistant to chemotherapy.Other references illustrating the use of retroviral vectors in genetherapy are: Clowes et al., J. Clin. Invest. 93:644-651 (1994); Kiem etal., Blood 83:1467-1473 (1994); Salmons and Gunzberg, Human Gene Therapy4:129-141 (1993); and Grossman and Wilson, Curr. Opin. in Genetics andDevel. 3:110-114 (1993). Lentiviral vectors contemplated for useinclude, for example, the HIV based vectors described in U.S. Pat. Nos.6,143,520; 5,665,557; and 5,981,276, which are herein incorporated byreference.

Adenoviruses are also contemplated for use in delivery of iRNAs.Adenoviruses are especially attractive vehicles, e.g., for deliveringgenes to respiratory epithelia. Adenoviruses naturally infectrespiratory epithelia where they cause a mild disease. Other targets foradenovirus-based delivery systems are liver, the central nervous system,endothelial cells, and muscle. Adenoviruses have the advantage of beingcapable of infecting non-dividing cells. Kozarsky and Wilson, CurrentOpinion in Genetics and Development 3:499-503 (1993) present a review ofadenovirus-based gene therapy. Bout et al., Human Gene Therapy 5:3-10(1994) demonstrated the use of adenovirus vectors to transfer genes tothe respiratory epithelia of rhesus monkeys. Other instances of the useof adenoviruses in gene therapy can be found in Rosenfeld et al.,Science 252:431-434 (1991); Rosenfeld et al., Cell 68:143-155 (1992);Mastrangeli et al., J. Clin. Invest. 91:225-234 (1993); PCT PublicationWO94/12649; and Wang, et al., Gene Therapy 2:775-783 (1995). A suitableAV vector for expressing an iRNA featured in the technology describedherein, a method for constructing the recombinant AV vector, and amethod for delivering the vector into target cells, are described in XiaH et al. (2002), Nat. Biotech. 20: 1006-1010.

Use of Adeno-associated virus (AAV) vectors is also contemplated (Walshet al., Proc. Soc. Exp. Biol. Med. 204:289-300 (1993); U.S. Pat. No.5,436,146). In one embodiment, the iRNA can be expressed as twoseparate, complementary single-stranded RNA molecules from a recombinantAAV vector having, for example, either the U6 or H1 RNA promoters, orthe cytomegalovirus (CMV) promoter. Suitable AAV vectors for expressingthe dsRNA featured in the technology described herein, methods forconstructing the recombinant AV vector, and methods for delivering thevectors into target cells are described in Samulski R et al. (1987), J.Virol. 61: 3096-3101; Fisher K J et al. (1996), J. Virol, 70: 520-532;Samulski R et al. (1989), J. Virol. 63: 3822-3826; U.S. Pat. No.5,252,479; U.S. Pat. No. 5,139,941; International Patent Application No.WO 94/13788; and International Patent Application No. WO 93/24641, theentire disclosures of which are herein incorporated by reference.

Another preferred viral vector is a pox virus such as a vaccinia virus,for example an attenuated vaccinia such as Modified Virus Ankara (MVA)or NYVAC, an avipox such as fowl pox or canary pox.

The tropism of viral vectors can be modified by pseudotyping the vectorswith envelope proteins or other surface antigens from other viruses, orby substituting different viral capsid proteins, as appropriate. Forexample, lentiviral vectors can be pseudotyped with surface proteinsfrom vesicular stomatitis virus (VSV), rabies, Ebola, Mokola, and thelike. AAV vectors can be made to target different cells by engineeringthe vectors to express different capsid protein serotypes; see, e.g.,Rabinowitz J E et al. (2002), J Virol 76:791-801, the entire disclosureof which is herein incorporated by reference.

The pharmaceutical preparation of a vector can include the vector in anacceptable diluent, or can include a slow release matrix in which thegene delivery vehicle is imbedded. Alternatively, where the completegene delivery vector can be produced intact from recombinant cells,e.g., retroviral vectors, the pharmaceutical preparation can include oneor more cells which produce the gene delivery system.

Pharmaceutical Compositions Containing iRNA

In one embodiment, provided herein are pharmaceutical compositionscontaining an iRNA, as described herein, and a pharmaceuticallyacceptable carrier. The pharmaceutical composition containing the iRNAis useful for treating a disease or disorder associated with theexpression or activity of a Serpina1 gene, e.g. alpha-1 anti-trypsindeficiency liver disease. Such pharmaceutical compositions areformulated based on the mode of delivery. One example is compositionsthat are formulated for systemic administration via parenteral delivery,e.g., by intravenous (IV) delivery. Another example is compositions thatare formulated for direct delivery into the brain parenchyma, e.g., byinfusion into the brain, such as by continuous pump infusion.

The pharmaceutical compositions featured herein are administered indosages sufficient to inhibit expression of Serpina1 genes. In general,a suitable dose of iRNA will be in the range of 0.001 to 200.0milligrams per kilogram body weight of the recipient per day, generallyin the range of 1 to 50 mg per kilogram body weight per day. Forexample, the dsRNA can be administered at 0.01 mg/kg, 0.05 mg/kg, 0.5mg/kg, 1 mg/kg, 1.5 mg/kg, 2 mg/kg, 3 mg/kg, 10 mg/kg, 20 mg/kg, 30mg/kg, 40 mg/kg, or 50 mg/kg per single dose. The pharmaceuticalcomposition can be administered once daily, or the iRNA can beadministered as two, three, or more sub-doses at appropriate intervalsthroughout the day or even using continuous infusion or delivery througha controlled release formulation. In that case, the iRNA contained ineach sub-dose must be correspondingly smaller in order to achieve thetotal daily dosage. The dosage unit can also be compounded for deliveryover several days, e.g., using a conventional sustained releaseformulation which provides sustained release of the iRNA over a severalday period. Sustained release formulations are well known in the art andare particularly useful for delivery of agents at a particular site,such as could be used with the agents of the technology describedherein. In this embodiment, the dosage unit contains a correspondingmultiple of the daily dose.

The effect of a single dose on Serpina1 levels can be long lasting, suchthat subsequent doses are administered at not more than 3, 4, or 5 dayintervals, or at not more than 1, 2, 3, or 4 week intervals.

The skilled artisan will appreciate that certain factors can influencethe dosage and timing required to effectively treat a subject, includingbut not limited to the severity of the disease or disorder, previoustreatments, the general health and/or age of the subject, and otherdiseases present. Moreover, treatment of a subject with atherapeutically effective amount of a composition can include a singletreatment or a series of treatments. Estimates of effective dosages andin vivo half-lives for the individual iRNAs encompassed by thetechnology described herein can be made using conventional methodologiesor on the basis of in vivo testing using an appropriate animal model, asdescribed elsewhere herein.

Advances in mouse genetics have generated a number of mouse models forthe study of various human diseases, such as pathological processesmediated by Serpina1 expression. Such models can be used for in vivotesting of iRNA, as well as for determining a therapeutically effectivedose. A suitable mouse model is, for example, a mouse containing atransgene expressing human Serpina1, e.g., an allele prone tomisfolding.

The technology described herein also includes pharmaceuticalcompositions and formulations which include the iRNA compounds featuredin the technology described herein. The pharmaceutical compositions ofthe technology described herein can be administered in a number of waysdepending upon whether local or systemic treatment is desired and uponthe area to be treated. Administration can be topical (e.g., by atransdermal patch), pulmonary, e.g., by inhalation or insufflation ofpowders or aerosols, including by nebulizer; intratracheal, intranasal,epidermal and transdermal, oral or parenteral. Parenteral administrationincludes intravenous, intraarterial, subcutaneous, intraperitoneal orintramuscular injection or infusion; subdermal, e.g., via an implanteddevice; or intracranial, e.g., by intraparenchymal, intrathecal orintraventricular, administration.

The iRNA can be delivered in a manner to target a particular tissue,such as the liver (e.g., the hepatocytes of the liver).

Pharmaceutical compositions and formulations for topical administrationcan include transdermal patches, ointments, lotions, creams, gels,drops, suppositories, sprays, liquids and powders. Conventionalpharmaceutical carriers, aqueous, powder or oily bases, thickeners andthe like can be necessary or desirable. Coated condoms, gloves and thelike can also be useful. Suitable topical formulations include those inwhich the iRNAs featured in the technology described herein are inadmixture with a topical delivery agent such as lipids, liposomes, fattyacids, fatty acid esters, steroids, chelating agents and surfactants.Suitable lipids and liposomes include neutral (e.g.,dioleoylphosphatidyl DOPE ethanolamine, dimyristoylphosphatidyl cholineDMPC, distearolyphosphatidyl choline) negative (e.g.,dimyristoylphosphatidyl glycerol DMPG) and cationic (e.g.,dioleoyltetramethylaminopropyl DOTAP and dioleoylphosphatidylethanolamine DOTMA). iRNAs featured in the technology described hereincan be encapsulated within liposomes or can form complexes thereto, inparticular to cationic liposomes. Alternatively, iRNAs can be complexedto lipids, in particular to cationic lipids. Suitable fatty acids andesters include but are not limited to arachidonic acid, oleic acid,eicosanoic acid, lauric acid, caprylic acid, capric acid, myristic acid,palmitic acid, stearic acid, linoleic acid, linolenic acid, dicaprate,tricaprate, monoolein, dilaurin, glyceryl 1-monocaprate,1-dodecylazacycloheptan-2-one, an acylcarnitine, an acylcholine, or aC₁₋₂₀ alkyl ester (e.g., isopropylmyristate IPM), monoglyceride,diglyceride or pharmaceutically acceptable salt thereof. Topicalformulations are described in detail in U.S. Pat. No. 6,747,014, whichis incorporated herein by reference.

Liposomal Formulations

There are many organized surfactant structures besides microemulsionsthat have been studied and used for the formulation of drugs. Theseinclude monolayers, micelles, bilayers and vesicles. Vesicles, such asliposomes, have attracted great interest because of their specificityand the duration of action they offer from the standpoint of drugdelivery. As used in the technology described herein, the term“liposome” means a vesicle composed of amphiphilic lipids arranged in aspherical bilayer or bilayers.

Liposomes are unilamellar or multilamellar vesicles which have amembrane formed from a lipophilic material and an aqueous interior. Theaqueous portion contains the composition to be delivered. Cationicliposomes possess the advantage of being able to fuse to the cell wall.Non-cationic liposomes, although not able to fuse as efficiently withthe cell wall, are taken up by macrophages in vivo.

In order to traverse intact mammalian skin, lipid vesicles must passthrough a series of fine pores, each with a diameter less than 50 nm,under the influence of a suitable transdermal gradient. Therefore, it isdesirable to use a liposome which is highly deformable and able to passthrough such fine pores.

Further advantages of liposomes include; liposomes obtained from naturalphospholipids are biocompatible and biodegradable; liposomes canincorporate a wide range of water and lipid soluble drugs; liposomes canprotect encapsulated drugs in their internal compartments frommetabolism and degradation (Rosoff, in Pharmaceutical Dosage Forms,Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., NewYork, N.Y., volume 1, p. 245). Important considerations in thepreparation of liposome formulations are the lipid surface charge,vesicle size and the aqueous volume of the liposomes.

Liposomes are useful for the transfer and delivery of active ingredientsto the site of action. Because the liposomal membrane is structurallysimilar to biological membranes, when liposomes are applied to a tissue,the liposomes start to merge with the cellular membranes and as themerging of the liposome and cell progresses, the liposomal contents areemptied into the cell where the active agent can act.

Liposomal formulations have been the focus of extensive investigation asthe mode of delivery for many drugs. There is growing evidence that fortopical administration, liposomes present several advantages over otherformulations. Such advantages include reduced side-effects related tohigh systemic absorption of the administered drug, increasedaccumulation of the administered drug at the desired target, and theability to administer a wide variety of drugs, both hydrophilic andhydrophobic, into the skin.

Several reports have detailed the ability of liposomes to deliver agentsincluding high-molecular weight nucleic acid into the skin. Compoundsincluding analgesics, antibodies, hormones and high-molecular weightDNAs have been administered to the skin. The majority of applicationsresulted in the targeting of the upper epidermis

Liposomes fall into two broad classes. Cationic liposomes are positivelycharged liposomes which interact with the negatively charged nucleicacid molecules to form a stable complex. The positively chargedDNA/liposome complex binds to the negatively charged cell surface and isinternalized in an endosome. Due to the acidic pH within the endosome,the liposomes are ruptured, releasing their contents into the cellcytoplasm (Wang et al., Biochem. Biophys. Res. Commun., 1987, 147,980-985).

Liposomes which are pH-sensitive or negatively-charged, entrap nucleicacids rather than complex with it. Since both the DNA and the lipid aresimilarly charged, repulsion rather than complex formation occurs.Nevertheless, some DNA is entrapped within the aqueous interior of theseliposomes. pH-sensitive liposomes have been used to deliver nucleicacids encoding the thymidine kinase gene to cell monolayers in culture.Expression of the exogenous gene was detected in the target cells (Zhouet al., Journal of Controlled Release, 1992, 19, 269-274).

One major type of liposomal composition includes phospholipids otherthan naturally-derived phosphatidylcholine. Neutral liposomecompositions, for example, can be formed from dimyristoylphosphatidylcholine (DMPC) or dipalmitoyl phosphatidylcholine (DPPC).Anionic liposome compositions generally are formed from dimyristoylphosphatidylglycerol, while anionic fusogenic liposomes are formedprimarily from dioleoyl phosphatidylethanolamine (DOPE). Another type ofliposomal composition is formed from phosphatidylcholine (PC) such as,for example, soybean PC, and egg PC. Another type is formed frommixtures of phospholipid and/or phosphatidylcholine and/or cholesterol.

Several studies have assessed the topical delivery of liposomal drugformulations to the skin. Application of liposomes containing interferonto guinea pig skin resulted in a reduction of skin herpes sores whiledelivery of interferon via other means (e.g., as a solution or as anemulsion) were ineffective (Weiner et al., Journal of Drug Targeting,1992, 2, 405-410). Further, an additional study tested the efficacy ofinterferon administered as part of a liposomal formulation to theadministration of interferon using an aqueous system, and concluded thatthe liposomal formulation was superior to aqueous administration (duPlessis et al., Antiviral Research, 1992, 18, 259-265).

Non-ionic liposomal systems have also been examined to determine theirutility in the delivery of drugs to the skin, in particular systemscomprising non-ionic surfactant and cholesterol. Non-ionic liposomalformulations comprising Novasome™ I (glyceryldilaurate/cholesterol/polyoxyethylene-10-stearyl ether) and Novasome™ II(glyceryl distearate/cholesterol/polyoxyethylene-10-stearyl ether) wereused to deliver cyclosporin-A into the dermis of mouse skin. Resultsindicated that such non-ionic liposomal systems were effective infacilitating the deposition of cyclosporine A into different layers ofthe skin (Hu et al. S.T.P.Pharma. Sci., 1994, 4(6) 466).

Liposomes also include “sterically stabilized” liposomes, a term which,as used herein, refers to liposomes comprising one or more specializedlipids that, when incorporated into liposomes, result in enhancedcirculation lifetimes relative to liposomes lacking such specializedlipids. Examples of sterically stabilized liposomes are those in whichpart of the vesicle-forming lipid portion of the liposome (A) comprisesone or more glycolipids, such as monosialoganglioside Gm′, or (B) isderivatized with one or more hydrophilic polymers, such as apolyethylene glycol (PEG) moiety. While not wishing to be bound by anyparticular theory, it is thought in the art that, at least forsterically stabilized liposomes containing gangliosides, sphingomyelin,or PEG-derivatized lipids, the enhanced circulation half-life of thesesterically stabilized liposomes derives from a reduced uptake into cellsof the reticuloendothelial system (RES) (Allen et al., FEBS Letters,1987, 223, 42; Wu et al., Cancer Research, 1993, 53, 3765).

Various liposomes comprising one or more glycolipids are known in theart. Papahadjopoulos et al. (Ann. N.Y. Acad. Sci., 1987, 507, 64)reported the ability of monosialoganglioside Gm′, galactocerebrosidesulfate and phosphatidylinositol to improve blood half-lives ofliposomes. These findings were expounded upon by Gabizon et al. (Proc.Natl. Acad. Sci. U.S.A., 1988, 85, 6949). U.S. Pat. No. 4,837,028 and WO88/04924, both to Allen et al., disclose liposomes comprising (1)sphingomyelin and (2) the ganglioside Gm′ or a galactocerebrosidesulfate ester. U.S. Pat. No. 5,543,152 (Webb et al.) discloses liposomescomprising sphingomyelin. Liposomes comprising1,2-sn-dimyristoylphosphatidylcholine are disclosed in WO 97/13499 (Limet al).

Many liposomes comprising lipids derivatized with one or morehydrophilic polymers, and methods of preparation thereof, are known inthe art. Sunamoto et al. (Bull. Chem. Soc. Jpn., 1980, 53, 2778)described liposomes comprising a nonionic detergent, 2C1215G, thatcontains a PEG moiety. Illum et al. (FEBS Lett., 1984, 167, 79) notedthat hydrophilic coating of polystyrene particles with polymeric glycolsresults in significantly enhanced blood half-lives. Syntheticphospholipids modified by the attachment of carboxylic groups ofpolyalkylene glycols (e.g., PEG) are described by Sears (U.S. Pat. Nos.4,426,330 and 4,534,899). Klibanov et al. (FEBS Lett., 1990, 268, 235)described experiments demonstrating that liposomes comprisingphosphatidylethanolamine (PE) derivatized with PEG or PEG stearate havesignificant increases in blood circulation half-lives. Blume et al.(Biochimica et Biophysica Acta, 1990, 1029, 91) extended suchobservations to other PEG-derivatized phospholipids, e.g., DSPE-PEG,formed from the combination of distearoylphosphatidylethanolamine (DSPE)and PEG. Liposomes having covalently bound PEG moieties on theirexternal surface are described in European Patent No. EP 0 445 131 B1and WO 90/04384 to Fisher. Liposome compositions containing 1-20 molepercent of PE derivatized with PEG, and methods of use thereof, aredescribed by Woodle et al. (U.S. Pat. Nos. 5,013,556 and 5,356,633) andMartin et al. (U.S. Pat. No. 5,213,804 and European Patent No. EP 0 496813 B1). Liposomes comprising a number of other lipid-polymer conjugatesare disclosed in WO 91/05545 and U.S. Pat. No. 5,225,212 (both to Martinet al.) and in WO 94/20073 (Zalipsky et al.) Liposomes comprisingPEG-modified ceramide lipids are described in WO 96/10391 (Choi et al).U.S. Pat. No. 5,540,935 (Miyazaki et al.) and U.S. Pat. No. 5,556,948(Tagawa et al.) describes PEG-containing liposomes that can be furtherderivatized with functional moieties on their surfaces.

A number of liposomes comprising nucleic acids are known in the art. WO96/40062 to Thierry et al. discloses methods for encapsulating highmolecular weight nucleic acids in liposomes. U.S. Pat. No. 5,264,221 toTagawa et al. discloses protein-bonded liposomes and asserts that thecontents of such liposomes can include a dsRNA. U.S. Pat. No. 5,665,710to Rahman et al. describes certain methods of encapsulatingoligodeoxynucleotides in liposomes. WO 97/04787 to Love et al. disclosesliposomes comprising dsRNAs targeted to the raf gene.

Transfersomes are yet another type of liposomes, and are highlydeformable lipid aggregates which are attractive candidates for drugdelivery vehicles. Transfersomes can be described as lipid dropletswhich are so highly deformable that they are easily able to penetratethrough pores which are smaller than the droplet. Transfersomes areadaptable to the environment in which they are used, e.g., they areself-optimizing (adaptive to the shape of pores in the skin),self-repairing, frequently reach their targets without fragmenting, andoften self-loading. To make transfersomes it is possible to add surfaceedge-activators, usually surfactants, to a standard liposomalcomposition. Transfersomes have been used to deliver serum albumin tothe skin. The transfersome-mediated delivery of serum albumin has beenshown to be as effective as subcutaneous injection of a solutioncontaining serum albumin.

Surfactants find wide application in formulations such as emulsions(including microemulsions) and liposomes. The most common way ofclassifying and ranking the properties of the many different types ofsurfactants, both natural and synthetic, is by the use of thehydrophile/lipophile balance (HLB). The nature of the hydrophilic group(also known as the “head”) provides the most useful means forcategorizing the different surfactants used in formulations (Rieger, inPharmaceutical Dosage Forms, Marcel Dekker, Inc., New York, N.Y., 1988,p. 285).

If the surfactant molecule is not ionized, it is classified as anonionic surfactant. Nonionic surfactants find wide application inpharmaceutical and cosmetic products and are usable over a wide range ofpH values. In general their HLB values range from 2 to about 18depending on their structure. Nonionic surfactants include nonionicesters such as ethylene glycol esters, propylene glycol esters, glycerylesters, polyglyceryl esters, sorbitan esters, sucrose esters, andethoxylated esters. Nonionic alkanolamides and ethers such as fattyalcohol ethoxylates, propoxylated alcohols, and ethoxylated/propoxylatedblock polymers are also included in this class. The polyoxyethylenesurfactants are the most popular members of the nonionic surfactantclass.

If the surfactant molecule carries a negative charge when it isdissolved or dispersed in water, the surfactant is classified asanionic. Anionic surfactants include carboxylates such as soaps, acyllactylates, acyl amides of amino acids, esters of sulfuric acid such asalkyl sulfates and ethoxylated alkyl sulfates, sulfonates such as alkylbenzene sulfonates, acyl isethionates, acyl taurates andsulfosuccinates, and phosphates. The most important members of theanionic surfactant class are the alkyl sulfates and the soaps.

If the surfactant molecule carries a positive charge when it isdissolved or dispersed in water, the surfactant is classified ascationic. Cationic surfactants include quaternary ammonium salts andethoxylated amines. The quaternary ammonium salts are the most usedmembers of this class.

If the surfactant molecule has the ability to carry either a positive ornegative charge, the surfactant is classified as amphoteric. Amphotericsurfactants include acrylic acid derivatives, substituted alkylamides,N-alkylbetaines and phosphatides.

The use of surfactants in drug products, formulations and in emulsionshas been reviewed (Rieger, in Pharmaceutical Dosage Forms, MarcelDekker, Inc., New York, N.Y., 1988, p. 285).

Nucleic Acid Lipid Particles

In one embodiment, a Serpina1 dsRNA featured in the technology describedherein is fully encapsulated in the lipid formulation, e.g., to form aSPLP, pSPLP, SNALP, or other nucleic acid-lipid particle. As usedherein, the term “SNALP” refers to a stable nucleic acid-lipid particle,including SPLP. As used herein, the term “SPLP” refers to a nucleicacid-lipid particle comprising plasmid DNA encapsulated within a lipidvesicle. SNALPs and SPLPs typically contain a cationic lipid, anon-cationic lipid, and a lipid that prevents aggregation of theparticle (e.g., a PEG-lipid conjugate). SNALPs and SPLPs are extremelyuseful for systemic applications, as they exhibit extended circulationlifetimes following intravenous (i.v.) injection and accumulate atdistal sites (e.g., sites physically separated from the administrationsite). SPLPs include “pSPLP,” which include an encapsulated condensingagent-nucleic acid complex as set forth in PCT Publication No. WO00/03683. The particles of the technology described herein typicallyhave a mean diameter of about 50 nm to about 150 nm, more typicallyabout 60 nm to about 130 nm, more typically about 70 nm to about 110 nm,most typically about 70 nm to about 90 nm, and are substantiallynontoxic. In addition, the nucleic acids when present in the nucleicacid-lipid particles of the technology described herein are resistant inaqueous solution to degradation with a nuclease. Nucleic acid-lipidparticles and their method of preparation are disclosed in, e.g., U.S.Pat. Nos. 5,976,567; 5,981,501; 6,534,484; 6,586,410; 6,815,432; and PCTPublication No. WO 96/40964.

In one embodiment, the lipid to drug ratio (mass/mass ratio) (e.g.,lipid to dsRNA ratio) will be in the range of from about 1:1 to about50:1, from about 1:1 to about 25:1, from about 3:1 to about 15:1, fromabout 4:1 to about 10:1, from about 5:1 to about 9:1, or about 6:1 toabout 9:1.

The cationic lipid can be, for example, N,N-dioleyl-N,N-dimethylammoniumchloride (DODAC), N,N-distearyl-N,N-dimethylammonium bromide (DDAB),N-(I-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTAP),N-(I-(2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTMA),N,N-dimethyl-2,3-dioleyloxy)propylamine (DODMA),1,2-DiLinoleyloxy-N,N-dimethylaminopropane (DLinDMA),1,2-Dilinolenyloxy-N,N-dimethylaminopropane (DLenDMA),1,2-Dilinoleylcarbamoyloxy-3-dimethylaminopropane (DLin-C-DAP),1,2-Dilinoleyoxy-3-(dimethylamino)acetoxypropane (DLin-DAC),1,2-Dilinoleyoxy-3-morpholinopropane (DLin-MA),1,2-Dilinoleoyl-3-dimethylaminopropane (DLinDAP),1,2-Dilinoleylthio-3-dimethylaminopropane (DLin-S-DMA),1-Linoleoyl-2-linoleyloxy-3-dimethylaminopropane (DLin-2-DMAP),1,2-Dilinoleyloxy-3-trimethylaminopropane chloride salt (DLin-TMA.Cl),1,2-Dilinoleoyl-3-trimethylaminopropane chloride salt (DLin-TAP.Cl),1,2-Dilinoleyloxy-3-(N-methylpiperazino)propane (DLin-MPZ), or3-(N,N-Dilinoleylamino)-1,2-propanediol (DLinAP),3-(N,N-Dioleylamino)-1,2-propanedio (DOAP),1,2-Dilinoleyloxo-3-(2-N,N-dimethylamino)ethoxypropane (DLin-EG-DMA),1,2-Dilinolenyloxy-N,N-dimethylaminopropane (DLinDMA),2,2-Dilinoleyl-4-dimethylaminomethyl[1,3]-dioxolane (DLin-K-DMA) oranalogs thereof,(3aR,5s,6aS)-N,N-dimethyl-2,2-di((9Z,12Z)-octadeca-9,12-dienyl)tetrahydro-3aH-cyclopenta[d][1,3]dioxol-5-amine(ALN100), (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl4-(dimethylamino)butanoate (MC3),1,1′-(2-(4-(2-((2-(bis(2-hydroxydodecyl)amino)ethyl)(2-hydroxydodecyl)amino)ethyl)piperazin-1-yl)ethylazanediyl)didodecan-2-ol(Tech G1), or a mixture thereof. The cationic lipid can comprise fromabout 20 mol % to about 50 mol % or about 40 mol % of the total lipidpresent in the particle.

In another embodiment, the compound2,2-Dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane can be used toprepare lipid-siRNA nanoparticles. Synthesis of2,2-Dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane is described in U.S.provisional patent application No. 61/107,998 filed on Oct. 23, 2008,which is herein incorporated by reference.

In one embodiment, the lipid-siRNA particle includes 40% 2,2-Dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane: 10% DSPC: 40%Cholesterol: 10% PEG-C-DOMG (mole percent) with a particle size of63.0±20 nm and a 0.027 siRNA/Lipid Ratio.

The ionizable/non-cationic lipid can be an anionic lipid or a neutrallipid including, but not limited to, distearoylphosphatidylcholine(DSPC), dioleoylphosphatidylcholine (DOPC),dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol(DOPG), dipalmitoylphosphatidylglycerol (DPPG),dioleoyl-phosphatidylethanolamine (DOPE),palmitoyloleoylphosphatidylcholine (POPC),palmitoyloleoylphosphatidylethanolamine (POPE),dioleoyl-phosphatidylethanolamine4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal), dipalmitoylphosphatidyl ethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE),distearoyl-phosphatidyl-ethanolamine (DSPE), 16-O-monomethyl PE,16-O-dimethyl PE, 18-1-trans PE,1-stearoyl-2-oleoyl-phosphatidyethanolamine (SOPE), cholesterol, or amixture thereof. The non-cationic lipid can be from about 5 mol % toabout 90 mol %, about 10 mol %, or about 58 mol % if cholesterol isincluded, of the total lipid present in the particle.

The conjugated lipid that inhibits aggregation of particles can be, forexample, a polyethyleneglycol (PEG)-lipid including, without limitation,a PEG-diacylglycerol (DAG), a PEG-dialkyloxypropyl (DAA), aPEG-phospholipid, a PEG-ceramide (Cer), or a mixture thereof. ThePEG-DAA conjugate can be, for example, a PEG-dilauryloxypropyl (Ci₂), aPEG-dimyristyloxypropyl (Ci₄), a PEG-dipalmityloxypropyl (Ci₆), or aPEG-distearyloxypropyl (Ci₈). The conjugated lipid that preventsaggregation of particles can be from 0 mol % to about 20 mol % or about2 mol % of the total lipid present in the particle.

In some embodiments, the nucleic acid-lipid particle further includescholesterol at, e.g., about 10 mol % to about 60 mol % or about 48 mol %of the total lipid present in the particle.

In one embodiment, the lipidoid ND98.4HCl (MW 1487) (see U.S. patentapplication Ser. No. 12/056,230, filed Mar. 26, 2008, which isincorporated herein by reference), Cholesterol (Sigma-Aldrich), andPEG-Ceramide C16 (Avanti Polar Lipids) can be used to preparelipid-dsRNA nanoparticles (i.e., LNP01 particles). Stock solutions ofeach in ethanol can be prepared as follows: ND98, 133 mg/ml;Cholesterol, 25 mg/ml, PEG-Ceramide C16, 100 mg/ml. The ND98,Cholesterol, and PEG-Ceramide C16 stock solutions can then be combinedin a, e.g., 42:48:10 molar ratio. The combined lipid solution can bemixed with aqueous dsRNA (e.g., in sodium acetate pH 5) such that thefinal ethanol concentration is about 35-45% and the final sodium acetateconcentration is about 100-300 mM. Lipid-dsRNA nanoparticles typicallyform spontaneously upon mixing. Depending on the desired particle sizedistribution, the resultant nanoparticle mixture can be extruded througha polycarbonate membrane (e.g., 100 nm cut-off) using, for example, athermobarrel extruder, such as Lipex Extruder (Northern Lipids, Inc). Insome cases, the extrusion step can be omitted. Ethanol removal andsimultaneous buffer exchange can be accomplished by, for example,dialysis or tangential flow filtration. Buffer can be exchanged with,for example, phosphate buffered saline (PBS) at about pH 7, e.g., aboutpH 6.9, about pH 7.0, about pH 7.1, about pH 7.2, about pH 7.3, or aboutpH 7.4.

LNP01 formulations are described, e.g., in International ApplicationPublication No. WO 2008/042973, which is hereby incorporated byreference.

Additional exemplary lipid-dsRNA formulations are as follows:

TABLE 1 cationic lipid/non-cationic lipid/cholesterol/PEG-lipidconjugate Ionizable/Cationic Lipid Lipid:siRNA ratio SNALP-11,2-Dilinolenyloxy-N,N-dimethylaminopropaneDLinDMA/DPPC/Cholesterol/PEG-cDMA (DLinDMA) (57.1/7.1/34.4/1.4)lipid:siRNA ~7:1 2-XTC 2,2-Dilinoleyl-4-dimethylaminoethyl-[1,3]-XTC/DPPC/Cholesterol/PEG-cDMA dioxolane (XTC) 57.1/7.1/34.4/1.4lipid:siRNA ~7:1 LNP05 2,2-Dilinoleyl-4-dimethylaminoethyl-[1,3]-XTC/DSPC/Cholesterol/PEG-DMG dioxolane (XTC) 57.5/7.5/31.5/3.5lipid:siRNA ~6:1 LNP06 2,2-Dilinoleyl-4-dimethylaminoethyl-[1,3]-XTC/DSPC/Cholesterol/PEG-DMG dioxolane (XTC) 57.5/7.5/31.5/3.5lipid:siRNA ~11:1 LNP07 2,2-Dilinoleyl-4-dimethylaminoethyl-[1,3]-XTC/DSPC/Cholesterol/PEG-DMG dioxolane (XTC) 60/7.5/31/1.5, lipid:siRNA~6:1 LNP08 2,2-Dilinoleyl-4-dimethylaminoethyl-[1,3]-XTC/DSPC/Cholesterol/PEG-DMG dioxolane (XTC) 60/7.5/31/1.5, lipid:siRNA~11:1 LNP09 2,2-Dilinoleyl-4-dimethylaminoethyl-[1,3]-XTC/DSPC/Cholesterol/PEG-DMG dioxolane (XTC) 50/10/38.5/1.5 Lipid:siRNA10:1 LNP10 (3aR,5s,6aS)-N,N-dimethyl-2,2-di((9Z,12Z)-ALN100/DSPC/Cholesterol/PEG-DMG octadeca-9,12-dienyl)tetrahydro-3aH-50/10/38.5/1.5 cyclopenta[d][1,3]dioxol-5-amine (ALN100) Lipid:siRNA10:1 LNP11 (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-MC-3/DSPC/Cholesterol/PEG-DMG tetraen-19-yl 4-(dimethylamino)butanoate50/10/38.5/1.5 (MC3) Lipid:siRNA 10:1 LNP12 1,1′-(2-(4-(2-((2-(bis(2-Tech G1/DSPC/Cholesterol/PEG-DMG hydroxydodecyl)amino)ethyl)(2-50/10/38.5/1.5 hydroxydodecyl)amino)ethyl)piperazin-1- Lipid:siRNA 10:1yl)ethylazanediyl)didodecan-2-ol (Tech G1) LNP13 XTCXTC/DSPC/Chol/PEG-DMG 50/10/38.5/1.5 Lipid:siRNA: 33:1 LNP14 MC3MC3/DSPC/Chol/PEG-DMG 40/15/40/5 Lipid:siRNA: 11:1 LNP15 MC3MC3/DSPC/Chol/PEG-DSG/GalNAc-PEG-DSG 50/10/35/4.5/0.5 Lipid:siRNA: 11:1LNP16 MC3 MC3/DSPC/Chol/PEG-DMG 50/10/38.5/1.5 Lipid:siRNA: 7:1 LNP17MC3 MC3/DSPC/Chol/PEG-DSG 50/10/38.5/1.5 Lipid:siRNA: 10:1 LNP18 MC3MC3/DSPC/Chol/PEG-DMG 50/10/38.5/1.5 Lipid:siRNA: 12:1 LNP19 MC3MC3/DSPC/Chol/PEG-DMG 50/10/35/5 Lipid:siRNA: 8:1 LNP20 MC3MC3/DSPC/Chol/PEG-DPG 50/10/38.5/1.5 Lipid:siRNA: 10:1 LNP21 C12-200C12-200/DSPC/Chol/PEG-DSG 50/10/38.5/1.5 Lipid:siRNA: 7:1 LNP22 XTCXTC/DSPC/Chol/PEG-DSG 50/10/38.5/1.5 Lipid:siRNA: 10:1

DSPC: distearoylphosphatidylcholine

DPPC: dipalmitoylphosphatidylcholine

PEG-DMG: PEG-didimyristoyl glycerol (C14-PEG, or PEG-C14) (PEG with avgmol wt of 2000)

PEG-DSG: PEG-distyryl glycerol (C18-PEG, or PEG-C18) (PEG with avg molwt of 2000)

PEG-cDMA: PEG-carbamoyl-1,2-dimyristyloxypropylamine (PEG with avg molwt of 2000)

SNALP (1,2-Dilinolenyloxy-N,N-dimethylaminopropane (DLinDMA)) comprisingformulations are described in International Publication No.WO2009/127060, filed Apr. 15, 2009, which is hereby incorporated byreference.

XTC comprising formulations are described, e.g., in U.S. ProvisionalSer. No. 61/148,366, filed Jan. 29, 2009; U.S. Provisional Ser. No.61/156,851, filed Mar. 2, 2009; U.S. Provisional Serial No. filed Jun.10, 2009; U.S. Provisional Ser. No. 61/228,373, filed Jul. 24, 2009;U.S. Provisional Ser. No. 61/239,686, filed Sep. 3, 2009, andInternational Application No. PCT/US2010/022614, filed Jan. 29, 2010,which are hereby incorporated by reference.

MC3 comprising formulations are described, e.g., in U.S. ProvisionalSer. No. 61/244,834, filed Sep. 22, 2009, U.S. Provisional Ser. No.61/185,800, filed Jun. 10, 2009, and International Application No.PCT/US10/28224, filed Jun. 10, 2010, which are hereby incorporated byreference.

ALNY-100 comprising formulations are described, e.g., Internationalpatent application number PCT/US09/63933, filed on Nov. 10, 2009, whichis hereby incorporated by reference.

C12-200 comprising formulations are described in U.S. Provisional Ser.No. 61/175,770, filed May 5, 2009 and International Application No.PCT/US10/33777, filed May 5, 2010, which are hereby incorporated byreference.

Synthesis of Ionizable/Cationic Lipids

Any of the compounds, e.g., cationic lipids and the like, used in thenucleic acid-lipid particles of the technology described herein can beprepared by known organic synthesis techniques, including the methodsdescribed in more detail in the Examples. All substituents are asdefined below unless indicated otherwise.

“Alkyl” means a straight chain or branched, noncyclic or cyclic,saturated aliphatic hydrocarbon containing from 1 to 24 carbon atoms.Representative saturated straight chain alkyls include methyl, ethyl,n-propyl, n-butyl, n-pentyl, n-hexyl, and the like; while saturatedbranched alkyls include isopropyl, sec-butyl, isobutyl, tert-butyl,isopentyl, and the like. Representative saturated cyclic alkyls includecyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and the like; whileunsaturated cyclic alkyls include cyclopentenyl and cyclohexenyl, andthe like.

“Alkenyl” means an alkyl, as defined above, containing at least onedouble bond between adjacent carbon atoms. Alkenyls include both cis andtrans isomers. Representative straight chain and branched alkenylsinclude ethylenyl, propylenyl, 1-butenyl, 2-butenyl, isobutylenyl,1-pentenyl, 2-pentenyl, 3-methyl-1-butenyl, 2-methyl-2-butenyl,2,3-dimethyl-2-butenyl, and the like.

“Alkynyl” means any alkyl or alkenyl, as defined above, whichadditionally contains at least one triple bond between adjacent carbons.Representative straight chain and branched alkynyls include acetylenyl,propynyl, 1-butynyl, 2-butynyl, 1-pentynyl, 2-pentynyl, 3-methyl-1butynyl, and the like.

“Acyl” means any alkyl, alkenyl, or alkynyl wherein the carbon at thepoint of attachment is substituted with an oxo group, as defined below.For example, —C(═O)alkyl, —C(═O)alkenyl, and —C(═O)alkynyl are acylgroups.

“Heterocycle” means a 5- to 7-membered monocyclic, or 7- to 10-memberedbicyclic, heterocyclic ring which is either saturated, unsaturated, oraromatic, and which contains from 1 or 2 heteroatoms independentlyselected from nitrogen, oxygen and sulfur, and wherein the nitrogen andsulfur heteroatoms can be optionally oxidized, and the nitrogenheteroatom can be optionally quaternized, including bicyclic rings inwhich any of the above heterocycles are fused to a benzene ring. Theheterocycle can be attached via any heteroatom or carbon atom.Heterocycles include heteroaryls as defined below. Heterocycles includemorpholinyl, pyrrolidinonyl, pyrrolidinyl, piperidinyl, piperizynyl,hydantoinyl, valerolactamyl, oxiranyl, oxetanyl, tetrahydrofuranyl,tetrahydropyranyl, tetrahydropyridinyl, tetrahydroprimidinyl,tetrahydrothiophenyl, tetrahydrothiopyranyl, tetrahydropyrimidinyl,tetrahydrothiophenyl, tetrahydrothiopyranyl, and the like.

The terms “optionally substituted alkyl”, “optionally substitutedalkenyl”, “optionally substituted alkynyl”, “optionally substitutedacyl”, and “optionally substituted heterocycle” means that, whensubstituted, at least one hydrogen atom is replaced with a substituent.In the case of an oxo substituent (═O) two hydrogen atoms are replaced.In this regard, substituents include oxo, halogen, heterocycle, —CN,—ORx, —NRxRy, —NRxC(═O)Ry, —NRxSO2Ry, —C(═O)Rx, —C(═O)ORx, —C(═O)NRxRy,—SOnRx and —SOnNRxRy, wherein n is 0, 1 or 2, Rx and Ry are the same ordifferent and independently hydrogen, alkyl or heterocycle, and each ofsaid alkyl and heterocycle substituents can be further substituted withone or more of oxo, halogen, —OH, —CN, alkyl, —ORx, heterocycle, —NRxRy,—NRxC(═O)Ry, —NRxSO2Ry, —C(═O)Rx, —C(═O)ORx, —C(═O)NRxRy, —SOnRx and—SOnNRxRy.

“Halogen” means fluoro, chloro, bromo and iodo.

In some embodiments, the methods of the technology described herein canrequire the use of protecting groups. Protecting group methodology iswell known to those skilled in the art (see, for example, ProtectiveGroups in Organic Synthesis, Green, T. W. et al., Wiley-Interscience,New York City, 1999). Briefly, protecting groups within the context ofthe technology described herein are any group that reduces or eliminatesunwanted reactivity of a functional group. A protecting group can beadded to a functional group to mask its reactivity during certainreactions and then removed to reveal the original functional group. Insome embodiments an “alcohol protecting group” is used. An “alcoholprotecting group” is any group which decreases or eliminates unwantedreactivity of an alcohol functional group. Protecting groups can beadded and removed using techniques well known in the art.

Synthesis of Formula A

In some embodiments, nucleic acid-lipid particles of the technologydescribed herein are formulated using a cationic lipid of formula A:

where R1 and R2 are independently alkyl, alkenyl or alkynyl, each can beoptionally substituted, and R3 and R4 are independently lower alkyl orR3 and R4 can be taken together to form an optionally substitutedheterocyclic ring. In some embodiments, the cationic lipid is XTC(2,2-Dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane). In general, thelipid of formula A above can be made by the following Reaction Schemes 1or 2, wherein all substituents are as defined above unless indicatedotherwise.

Lipid A, where R1 and R2 are independently alkyl, alkenyl or alkynyl,each can be optionally substituted, and R3 and R4 are independentlylower alkyl or R3 and R4 can be taken together to form an optionallysubstituted heterocyclic ring, can be prepared according to Scheme 1.Ketone 1 and bromide 2 can be purchased or prepared according to methodsknown to those of ordinary skill in the art. Reaction of 1 and 2 yieldsketal 3. Treatment of ketal 3 with amine 4 yields lipids of formula A.The lipids of formula A can be converted to the corresponding ammoniumsalt with an organic salt of formula 5, where X is anion counter ionselected from halogen, hydroxide, phosphate, sulfate, or the like.

Alternatively, the ketone 1 starting material can be prepared accordingto Scheme 2. Grignard reagent 6 and cyanide 7 can be purchased orprepared according to methods known to those of ordinary skill in theart. Reaction of 6 and 7 yields ketone 1. Conversion of ketone 1 to thecorresponding lipids of formula A is as described in Scheme 1.

Synthesis of MC3

Preparation of DLin-M-C3-DMA (i.e.,(6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl4-(dimethylamino)butanoate) was as follows. A solution of(6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-ol (0.53 g),4-N,N-dimethylaminobutyric acid hydrochloride (0.51 g),4-N,N-dimethylaminopyridine (0.61 g) and1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (0.53 g) indichloromethane (5 mL) was stirred at room temperature overnight. Thesolution was washed with dilute hydrochloric acid followed by diluteaqueous sodium bicarbonate. The organic fractions were dried overanhydrous magnesium sulphate, filtered and the solvent removed on arotovap. The residue was passed down a silica gel column (20 g) using a1-5% methanol/dichloromethane elution gradient. Fractions containing thepurified product were combined and the solvent removed, yielding acolorless oil (0.54 g).

Synthesis of ALNY-100

Synthesis of ketal 519 [ALNY-100] was performed using the followingscheme 3:

Synthesis of 515

To a stirred suspension of LiAlH4 (3.74 g, 0.09852 mol) in 200 mlanhydrous THF in a two neck RBF (1 L), was added a solution of 514 (10g, 0.04926 mol) in 70 mL of THF slowly at 0° C. under nitrogenatmosphere. After complete addition, reaction mixture was warmed to roomtemperature and then heated to reflux for 4 h. Progress of the reactionwas monitored by TLC. After completion of reaction (by TLC) the mixturewas cooled to 0° C. and quenched with careful addition of saturatedNa2SO4 solution. Reaction mixture was stirred for 4 h at roomtemperature and filtered off. Residue was washed well with THF. Thefiltrate and washings were mixed and diluted with 400 mL dioxane and 26mL conc. HCl and stirred for 20 minutes at room temperature. Thevolatilities were stripped off under vacuum to furnish the hydrochloridesalt of 515 as a white solid. Yield: 7.12 g 1H-NMR (DMSO, 400 MHz):δ=9.34 (broad, 2H), 5.68 (s, 2H), 3.74 (m, 1H), 2.66-2.60 (m, 2H),2.50-2.45 (m, 5H).

Synthesis of 516

To a stirred solution of compound 515 in 100 mL dry DCM in a 250 mL twoneck RBF, was added NEt3 (37.2 mL, 0.2669 mol) and cooled to 0° C. undernitrogen atmosphere. After a slow addition ofN-(benzyloxy-carbonyloxy)-succinimide (20 g, 0.08007 mol) in 50 mL dryDCM, reaction mixture was allowed to warm to room temperature. Aftercompletion of the reaction (2-3 h by TLC) mixture was washedsuccessively with 1N HCl solution (1×100 mL) and saturated NaHCO3solution (1×50 mL). The organic layer was then dried over anhyd. Na2SO4and the solvent was evaporated to give crude material which was purifiedby silica gel column chromatography to get 516 as sticky mass. Yield: 11g (89%). 1H-NMR (CDCl3, 400 MHz): δ=7.36-7.27 (m, 5H), 5.69 (s, 2H),5.12 (s, 2H), 4.96 (br., 1H) 2.74 (s, 3H), 2.60 (m, 2H), 2.30-2.25 (m,2H). LC-MS [M+H]-232.3 (96.94%).

Synthesis of 517A and 517B

The cyclopentene 516 (5 g, 0.02164 mol) was dissolved in a solution of220 mL acetone and water (10:1) in a single neck 500 mL RBF and to itwas added N-methyl morpholine-N-oxide (7.6 g, 0.06492 mol) followed by4.2 mL of 7.6% solution of OsO4 (0.275 g, 0.00108 mol) in tert-butanolat room temperature. After completion of the reaction (˜3 h), themixture was quenched with addition of solid Na2SO3 and resulting mixturewas stirred for 1.5 h at room temperature. Reaction mixture was dilutedwith DCM (300 mL) and washed with water (2×100 mL) followed by saturatedNaHCO3 (1×50 mL) solution, water (1×30 mL) and finally with brine (lx 50mL). Organic phase was dried over an.Na2SO4 and solvent was removed invacuum. Silica gel column chromatographic purification of the crudematerial was afforded a mixture of diastereomers, which were separatedby prep HPLC. Yield: −6 g crude

517A—Peak-1 (white solid), 5.13 g (96%). 1H-NMR (DMSO, 400 MHz):δ=7.39-7.31 (m, 5H), 5.04 (s, 2H), 4.78-4.73 (m, 1H), 4.48-4.47 (d, 2H),3.94-3.93 (m, 2H), 2.71 (s, 3H), 1.72-1.67 (m, 4H). LC-MS—[M+H]-266.3,[M+NH4+]-283.5 present, HPLC-97.86%. Stereochemistry confirmed by X-ray.

Synthesis of 518

Using a procedure analogous to that described for the synthesis ofcompound 505, compound 518 (1.2 g, 41%) was obtained as a colorless oil.1H-NMR (CDCl3, 400 MHz): δ=7.35-7.33 (m, 4H), 7.30-7.27 (m, 1H),5.37-5.27 (m, 8H), 5.12 (s, 2H), 4.75 (m, 1H), 4.58-4.57 (m, 2H),2.78-2.74 (m, 7H), 2.06-2.00 (m, 8H), 1.96-1.91 (m, 2H), 1.62 (m, 4H),1.48 (m, 2H), 1.37-1.25 (br m, 36H), 0.87 (m, 6H). HPLC-98.65%.

General Procedure for the Synthesis of Compound 519

A solution of compound 518 (1 eq) in hexane (15 mL) was added in adrop-wise fashion to an ice-cold solution of LAH in THF (1 M, 2 eq).After complete addition, the mixture was heated at 40° C. over 0.5 hthen cooled again on an ice bath. The mixture was carefully hydrolyzedwith saturated aqueous Na2SO4 then filtered through celite and reducedto an oil. Column chromatography provided the pure 519 (1.3 g, 68%)which was obtained as a colorless oil. 13C NMR □=130.2, 130.1 (×2),127.9 (×3), 112.3, 79.3, 64.4, 44.7, 38.3, 35.4, 31.5, 29.9 (×2), 29.7,29.6 (×2), 29.5 (×3), 29.3 (×2), 27.2 (×3), 25.6, 24.5, 23.3, 226, 14.1;Electrospray MS (+ve): Molecular weight for C44H80NO2 (M+H)+ Calc.654.6, Found 654.6.

Formulations prepared by either the standard or extrusion-free methodcan be characterized in similar manners. For example, formulations aretypically characterized by visual inspection. They should be whitishtranslucent solutions free from aggregates or sediment. Particle sizeand particle size distribution of lipid-nanoparticles can be measured bylight scattering using, for example, a Malvern Zetasizer Nano ZS(Malvern, USA). Particles should be about 20-300 nm, such as 40-100 nmin size. The particle size distribution should be unimodal. The totaldsRNA concentration in the formulation, as well as the entrappedfraction, is estimated using a dye exclusion assay. A sample of theformulated dsRNA can be incubated with an RNA-binding dye, such asRibogreen (Molecular Probes) in the presence or absence of a formulationdisrupting surfactant, e.g., 0.5% Triton-X100. The total dsRNA in theformulation can be determined by the signal from the sample containingthe surfactant, relative to a standard curve. The entrapped fraction isdetermined by subtracting the “free” dsRNA content (as measured by thesignal in the absence of surfactant) from the total dsRNA content.Percent entrapped dsRNA is typically >85%. For SNALP formulation, theparticle size is at least 30 nm, at least 40 nm, at least 50 nm, atleast 60 nm, at least 70 nm, at least 80 nm, at least 90 nm, at least100 nm, at least 110 nm, and at least 120 nm. The suitable range istypically about at least 50 nm to about at least 110 nm, about at least60 nm to about at least 100 nm, or about at least 80 nm to about atleast 90 nm.

Compositions and formulations for oral administration include powders orgranules, microparticulates, nanoparticulates, suspensions or solutionsin water or non-aqueous media, capsules, gel capsules, sachets, tabletsor minitablets. Thickeners, flavoring agents, diluents, emulsifiers,dispersing aids or binders can be desirable. In some embodiments, oralformulations are those in which dsRNAs featured in the technologydescribed herein are administered in conjunction with one or morepenetration enhancer surfactants and chelators. Suitable surfactantsinclude fatty acids and/or esters or salts thereof, bile acids and/orsalts thereof. Suitable bile acids/salts include chenodeoxycholic acid(CDCA) and ursodeoxychenodeoxycholic acid (UDCA), cholic acid,dehydrocholic acid, deoxycholic acid, glucholic acid, glycholic acid,glycodeoxycholic acid, taurocholic acid, taurodeoxycholic acid, sodiumtauro-24,25-dihydro-fusidate and sodium glycodihydrofusidate. Suitablefatty acids include arachidonic acid, undecanoic acid, oleic acid,lauric acid, caprylic acid, capric acid, myristic acid, palmitic acid,stearic acid, linoleic acid, linolenic acid, dicaprate, tricaprate,monoolein, dilaurin, glyceryl 1-monocaprate,1-dodecylazacycloheptan-2-one, an acylcarnitine, an acylcholine, or amonoglyceride, a diglyceride or a pharmaceutically acceptable saltthereof (e.g., sodium). In some embodiments, combinations of penetrationenhancers are used, for example, fatty acids/salts in combination withbile acids/salts. One exemplary combination is the sodium salt of lauricacid, capric acid and UDCA. Further penetration enhancers includepolyoxyethylene-9-lauryl ether, polyoxyethylene-20-cetyl ether. DsRNAsfeatured in the technology described herein can be delivered orally, ingranular form including sprayed dried particles, or complexed to formmicro or nanoparticles. DsRNA complexing agents include poly-aminoacids; polyimines; polyacrylates; polyalkylacrylates, polyoxethanes,polyalkylcyanoacrylates; cationized gelatins, albumins, starches,acrylates, polyethyleneglycols (PEG) and starches;polyalkylcyanoacrylates; DEAE-derivatized polyimines, pollulans,celluloses and starches. Suitable complexing agents include chitosan,N-trimethylchitosan, poly-L-lysine, polyhistidine, polyornithine,polyspermines, protamine, polyvinylpyridine,polythiodiethylaminomethylethylene P(TDAE), polyaminostyrene (e.g.,p-amino), poly(methylcyanoacrylate), poly(ethylcyanoacrylate),poly(butylcyanoacrylate), poly(isobutylcyanoacrylate),poly(isohexylcynaoacrylate), DEAE-methacrylate, DEAE-hexylacrylate,DEAE-acrylamide, DEAE-albumin and DEAE-dextran, polymethylacrylate,polyhexylacrylate, poly(D,L-lactic acid), poly(DL-lactic-co-glycolicacid (PLGA), alginate, and polyethyleneglycol (PEG). Oral formulationsfor dsRNAs and their preparation are described in detail in U.S. Pat.No. 6,887,906, US Publn. No. 20030027780, and U.S. Pat. No. 6,747,014,each of which is incorporated herein by reference.

Compositions and formulations for parenteral, intraparenchymal (into thebrain), intrathecal, intraventricular or intrahepatic administration caninclude sterile aqueous solutions which can also contain buffers,diluents and other suitable additives such as, but not limited to,penetration enhancers, carrier compounds and other pharmaceuticallyacceptable carriers or excipients.

Pharmaceutical compositions of the technology described herein include,but are not limited to, solutions, emulsions, and liposome-containingformulations. These compositions can be generated from a variety ofcomponents that include, but are not limited to, preformed liquids,self-emulsifying solids and self-emulsifying semisolids. Particularlypreferred are formulations that target the liver when treating hepaticdisorders such as hepatic carcinoma.

The pharmaceutical formulations of the technology described herein,which can conveniently be presented in unit dosage form, can be preparedaccording to conventional techniques well known in the pharmaceuticalindustry. Such techniques include the step of bringing into associationthe active ingredients with the pharmaceutical carrier(s) orexcipient(s). In general, the formulations are prepared by uniformly andintimately bringing into association the active ingredients with liquidcarriers or finely divided solid carriers or both, and then, ifnecessary, shaping the product.

The compositions of the technology described herein can be formulatedinto any of many possible dosage forms such as, but not limited to,tablets, capsules, gel capsules, liquid syrups, soft gels,suppositories, and enemas. The compositions of the technology describedherein can also be formulated as suspensions in aqueous, non-aqueous ormixed media. Aqueous suspensions can further contain substances whichincrease the viscosity of the suspension including, for example, sodiumcarboxymethylcellulose, sorbitol and/or dextran. The suspension can alsocontain stabilizers.

Additional Formulations Emulsions

The compositions of the technology described herein can be prepared andformulated as emulsions. Emulsions are typically heterogeneous systemsof one liquid dispersed in another in the form of droplets usuallyexceeding 0.1 μm in diameter (see e.g., Ansel's Pharmaceutical DosageForms and Drug Delivery Systems, Allen, L V., Popovich N G., and Ansel HC., 2004, Lippincott Williams & Wilkins (8th ed.), New York, N.Y.;Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker(Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199;Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker(Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., Volume 1, p. 245;Block in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker(Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 2, p. 335;Higuchi et al., in Remington's Pharmaceutical Sciences, Mack PublishingCo., Easton, Pa., 1985, p. 301). Emulsions are often biphasic systemscomprising two immiscible liquid phases intimately mixed and dispersedwith each other. In general, emulsions can be of either the water-in-oil(w/o) or the oil-in-water (o/w) variety. When an aqueous phase is finelydivided into and dispersed as minute droplets into a bulk oily phase,the resulting composition is called a water-in-oil (w/o) emulsion.Alternatively, when an oily phase is finely divided into and dispersedas minute droplets into a bulk aqueous phase, the resulting compositionis called an oil-in-water (o/w) emulsion. Emulsions can containadditional components in addition to the dispersed phases, and theactive drug which can be present as a solution in either the aqueousphase, oily phase or itself as a separate phase. Pharmaceuticalexcipients such as emulsifiers, stabilizers, dyes, and anti-oxidants canalso be present in emulsions as needed. Pharmaceutical emulsions canalso be multiple emulsions that are comprised of more than two phasessuch as, for example, in the case of oil-in-water-in-oil (o/w/o) andwater-in-oil-in-water (w/o/w) emulsions. Such complex formulations oftenprovide certain advantages that simple binary emulsions do not. Multipleemulsions in which individual oil droplets of an o/w emulsion enclosesmall water droplets constitute a w/o/w emulsion. Likewise a system ofoil droplets enclosed in globules of water stabilized in an oilycontinuous phase provides an o/w/o emulsion.

Emulsions are characterized by little or no thermodynamic stability.Often, the dispersed or discontinuous phase of the emulsion is welldispersed into the external or continuous phase and maintained in thisform through the means of emulsifiers or the viscosity of theformulation. Either of the phases of the emulsion can be a semisolid ora solid, as is the case of emulsion-style ointment bases and creams.Other means of stabilizing emulsions entail the use of emulsifiers thatcan be incorporated into either phase of the emulsion. Emulsifiers canbroadly be classified into four categories: synthetic surfactants,naturally occurring emulsifiers, absorption bases, and finely dispersedsolids (see e.g., Ansel's Pharmaceutical Dosage Forms and Drug DeliverySystems, Allen, L V., Popovich N G., and Ansel H C., 2004, LippincottWilliams & Wilkins (8th ed.), New York, N.Y.; Idson, in PharmaceuticalDosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker,Inc., New York, N.Y., volume 1, p. 199).

Synthetic surfactants, also known as surface active agents, have foundwide applicability in the formulation of emulsions and have beenreviewed in the literature (see e.g., Ansel's Pharmaceutical DosageForms and Drug Delivery Systems, Allen, L V., Popovich N G., and Ansel HC., 2004, Lippincott Williams & Wilkins (8th ed.), New York, N.Y.;Rieger, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker(Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 285;Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker(Eds.), Marcel Dekker, Inc., New York, N.Y., 1988, volume 1, p. 199).Surfactants are typically amphiphilic and comprise a hydrophilic and ahydrophobic portion. The ratio of the hydrophilic to the hydrophobicnature of the surfactant has been termed the hydrophile/lipophilebalance (HLB) and is a valuable tool in categorizing and selectingsurfactants in the preparation of formulations. Surfactants can beclassified into different classes based on the nature of the hydrophilicgroup: nonionic, anionic, cationic and amphoteric (see e.g., Ansel'sPharmaceutical Dosage Forms and Drug Delivery Systems, Allen, L V.,Popovich N G., and Ansel H C., 2004, Lippincott Williams & Wilkins (8thed.), New York, N.Y. Rieger, in Pharmaceutical Dosage Forms, Lieberman,Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y.,volume 1, p. 285).

Naturally occurring emulsifiers used in emulsion formulations includelanolin, beeswax, phosphatides, lecithin and acacia. Absorption basespossess hydrophilic properties such that they can soak up water to formw/o emulsions yet retain their semisolid consistencies, such asanhydrous lanolin and hydrophilic petrolatum. Finely divided solids havealso been used as good emulsifiers especially in combination withsurfactants and in viscous preparations. These include polar inorganicsolids, such as heavy metal hydroxides, nonswelling clays such asbentonite, attapulgite, hectorite, kaolin, montmorillonite, colloidalaluminum silicate and colloidal magnesium aluminum silicate, pigmentsand nonpolar solids such as carbon or glyceryl tristearate.

A large variety of non-emulsifying materials are also included inemulsion formulations and contribute to the properties of emulsions.These include fats, oils, waxes, fatty acids, fatty alcohols, fattyesters, humectants, hydrophilic colloids, preservatives and antioxidants(Block, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker(Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 335;Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker(Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199).

Hydrophilic colloids or hydrocolloids include naturally occurring gumsand synthetic polymers such as polysaccharides (for example, acacia,agar, alginic acid, carrageenan, guar gum, karaya gum, and tragacanth),cellulose derivatives (for example, carboxymethylcellulose andcarboxypropylcellulose), and synthetic polymers (for example, carbomers,cellulose ethers, and carboxyvinyl polymers). These disperse or swell inwater to form colloidal solutions that stabilize emulsions by formingstrong interfacial films around the dispersed-phase droplets and byincreasing the viscosity of the external phase.

Since emulsions often contain a number of ingredients such ascarbohydrates, proteins, sterols and phosphatides that can readilysupport the growth of microbes, these formulations often incorporatepreservatives. Commonly used preservatives included in emulsionformulations include methyl paraben, propyl paraben, quaternary ammoniumsalts, benzalkonium chloride, esters of p-hydroxybenzoic acid, and boricacid. Antioxidants are also commonly added to emulsion formulations toprevent deterioration of the formulation. Antioxidants used can be freeradical scavengers such as tocopherols, alkyl gallates, butylatedhydroxyanisole, butylated hydroxytoluene, or reducing agents such asascorbic acid and sodium metabisulfite, and antioxidant synergists suchas citric acid, tartaric acid, and lecithin.

The application of emulsion formulations via dermatological, oral andparenteral routes and methods for their manufacture have been reviewedin the literature (see e.g., Ansel's Pharmaceutical Dosage Forms andDrug Delivery Systems, Allen, L V., Popovich N G., and Ansel H C., 2004,Lippincott Williams & Wilkins (8th ed.), New York, N.Y.; Idson, inPharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988,Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199). Emulsionformulations for oral delivery have been very widely used because ofease of formulation, as well as efficacy from an absorption andbioavailability standpoint (see e.g., Ansel's Pharmaceutical DosageForms and Drug Delivery Systems, Allen, L V., Popovich N G., and Ansel HC., 2004, Lippincott Williams & Wilkins (8th ed.), New York, N.Y.;Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker(Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 245;Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker(Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199).Mineral-oil base laxatives, oil-soluble vitamins and high fat nutritivepreparations are among the materials that have commonly beenadministered orally as o/w emulsions.

In one embodiment of the technology described herein, the compositionsof iRNAs and nucleic acids are formulated as microemulsions. Amicroemulsion can be defined as a system of water, oil and amphiphilewhich is a single optically isotropic and thermodynamically stableliquid solution (see e.g., Ansel's Pharmaceutical Dosage Forms and DrugDelivery Systems, Allen, L V., Popovich N G., and Ansel H C., 2004,Lippincott Williams & Wilkins (8th ed.), New York, N.Y.; Rosoff, inPharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988,Marcel Dekker, Inc., New York, N.Y., volume 1, p. 245). Typicallymicroemulsions are systems that are prepared by first dispersing an oilin an aqueous surfactant solution and then adding a sufficient amount ofa fourth component, generally an intermediate chain-length alcohol toform a transparent system. Therefore, microemulsions have also beendescribed as thermodynamically stable, isotropically clear dispersionsof two immiscible liquids that are stabilized by interfacial films ofsurface-active molecules (Leung and Shah, in: Controlled Release ofDrugs: Polymers and Aggregate Systems, Rosoff, M., Ed., 1989, VCHPublishers, New York, pages 185-215). Microemulsions commonly areprepared via a combination of three to five components that include oil,water, surfactant, cosurfactant and electrolyte. Whether themicroemulsion is of the water-in-oil (w/o) or an oil-in-water (o/w) typeis dependent on the properties of the oil and surfactant used and on thestructure and geometric packing of the polar heads and hydrocarbon tailsof the surfactant molecules (Schott, in Remington's PharmaceuticalSciences, Mack Publishing Co., Easton, Pa., 1985, p. 271).

The phenomenological approach utilizing phase diagrams has beenextensively studied and has yielded a comprehensive knowledge, to oneskilled in the art, of how to formulate microemulsions (see e.g.,Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, LV., Popovich N G., and Ansel H C., 2004, Lippincott Williams & Wilkins(8th ed.), New York, N.Y.; Rosoff, in Pharmaceutical Dosage Forms,Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., NewYork, N.Y., volume 1, p. 245; Block, in Pharmaceutical Dosage Forms,Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., NewYork, N.Y., volume 1, p. 335). Compared to conventional emulsions,microemulsions offer the advantage of solubilizing water-insoluble drugsin a formulation of thermodynamically stable droplets that are formedspontaneously.

Surfactants used in the preparation of microemulsions include, but arenot limited to, ionic surfactants, non-ionic surfactants, Brij 96,polyoxyethylene oleyl ethers, polyglycerol fatty acid esters,tetraglycerol monolaurate (ML310), tetraglycerol monooleate (MO310),hexaglycerol monooleate (PO310), hexaglycerol pentaoleate (PO500),decaglycerol monocaprate (MCA750), decaglycerol monooleate (MO750),decaglycerol sequioleate (SO750), decaglycerol decaoleate (DA0750),alone or in combination with cosurfactants. The cosurfactant, usually ashort-chain alcohol such as ethanol, 1-propanol, and 1-butanol, servesto increase the interfacial fluidity by penetrating into the surfactantfilm and consequently creating a disordered film because of the voidspace generated among surfactant molecules. Microemulsions can, however,be prepared without the use of cosurfactants and alcohol-freeself-emulsifying microemulsion systems are known in the art. The aqueousphase can typically be, but is not limited to, water, an aqueoussolution of the drug, glycerol, PEG300, PEG400, polyglycerols, propyleneglycols, and derivatives of ethylene glycol. The oil phase can include,but is not limited to, materials such as Captex 300, Captex 355, CapmulMCM, fatty acid esters, medium chain (C8-C12) mono, di, andtri-glycerides, polyoxyethylated glyceryl fatty acid esters, fattyalcohols, polyglycolized glycerides, saturated polyglycolized C8-C10glycerides, vegetable oils and silicone oil.

Microemulsions are particularly of interest from the standpoint of drugsolubilization and the enhanced absorption of drugs. Lipid basedmicroemulsions (both o/w and w/o) have been proposed to enhance the oralbioavailability of drugs, including peptides (see e.g., U.S. Pat. Nos.6,191,105; 7,063,860; 7,070,802; 7,157,099; Constantinides et al.,Pharmaceutical Research, 1994, 11, 1385-1390; Ritschel, Meth. Find. Exp.Clin. Pharmacol., 1993, 13, 205). Microemulsions afford advantages ofimproved drug solubilization, protection of drug from enzymatichydrolysis, possible enhancement of drug absorption due tosurfactant-induced alterations in membrane fluidity and permeability,ease of preparation, ease of oral administration over solid dosageforms, improved clinical potency, and decreased toxicity (see e.g., U.S.Pat. Nos. 6,191,105; 7,063,860; 7,070,802; 7,157,099; Constantinides etal., Pharmaceutical Research, 1994, 11, 1385; Ho et al., J. Pharm. Sci.,1996, 85, 138-143). Often microemulsions can form spontaneously whentheir components are brought together at ambient temperature. This canbe particularly advantageous when formulating thermolabile drugs,peptides or iRNAs. Microemulsions have also been effective in thetransdermal delivery of active components in both cosmetic andpharmaceutical applications. It is expected that the microemulsioncompositions and formulations of the technology described herein willfacilitate the increased systemic absorption of iRNAs and nucleic acidsfrom the gastrointestinal tract, as well as improve the local cellularuptake of iRNAs and nucleic acids.

Microemulsions of the technology described herein can also containadditional components and additives such as sorbitan monostearate (Grill3), Labrasol, and penetration enhancers to improve the properties of theformulation and to enhance the absorption of the iRNAs and nucleic acidsof the technology described herein. Penetration enhancers used in themicroemulsions of the technology described herein can be classified asbelonging to one of five broad categories—surfactants, fatty acids, bilesalts, chelating agents, and non-chelating non-surfactants (Lee et al.,Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p. 92). Eachof these classes has been discussed above.

Penetration Enhancers

In one embodiment, the technology described herein employs variouspenetration enhancers to effect the efficient delivery of nucleic acids,particularly iRNAs, to the skin of animals. Most drugs are present insolution in both ionized and nonionized forms. However, usually onlylipid soluble or lipophilic drugs readily cross cell membranes. It hasbeen discovered that even non-lipophilic drugs can cross cell membranesif the membrane to be crossed is treated with a penetration enhancer. Inaddition to aiding the diffusion of non-lipophilic drugs across cellmembranes, penetration enhancers also enhance the permeability oflipophilic drugs.

Penetration enhancers can be classified as belonging to one of fivebroad categories, i.e., surfactants, fatty acids, bile salts, chelatingagents, and non-chelating non-surfactants (see e.g., Malmsten, M.Surfactants and polymers in drug delivery, Informa Health Care, NewYork, N.Y., 2002; Lee et al., Critical Reviews in Therapeutic DrugCarrier Systems, 1991, p. 92). Each of the above mentioned classes ofpenetration enhancers are described below in greater detail.

Surfactants: In connection with the technology described herein,surfactants (or “surface-active agents”) are chemical entities which,when dissolved in an aqueous solution, reduce the surface tension of thesolution or the interfacial tension between the aqueous solution andanother liquid, with the result that absorption of iRNAs through themucosa is enhanced. In addition to bile salts and fatty acids, thesepenetration enhancers include, for example, sodium lauryl sulfate,polyoxyethylene-9-lauryl ether and polyoxyethylene-20-cetyl ether) (seee.g., Malmsten, M. Surfactants and polymers in drug delivery, InformaHealth Care, New York, N.Y., 2002; Lee et al., Critical Reviews inTherapeutic Drug Carrier Systems, 1991, p. 92); and perfluorochemicalemulsions, such as FC-43. Takahashi et al., J. Pharm. Pharmacol., 1988,40, 252).

Fatty acids: Various fatty acids and their derivatives which act aspenetration enhancers include, for example, oleic acid, lauric acid,capric acid (n-decanoic acid), myristic acid, palmitic acid, stearicacid, linoleic acid, linolenic acid, dicaprate, tricaprate, monoolein(1-monooleoyl-rac-glycerol), dilaurin, caprylic acid, arachidonic acid,glycerol 1-monocaprate, 1-dodecylazacycloheptan-2-one, acylcarnitines,acylcholines, C1-20 alkyl esters thereof (e.g., methyl, isopropyl andt-butyl), and mono- and di-glycerides thereof (i.e., oleate, laurate,caprate, myristate, palmitate, stearate, linoleate, etc.) (see e.g.,Touitou, E., et al. Enhancement in Drug Delivery, CRC Press, Danvers,Mass., 2006; Lee et al., Critical Reviews in Therapeutic Drug CarrierSystems, 1991, p. 92; Muranishi, Critical Reviews in Therapeutic DrugCarrier Systems, 1990, 7, 1-33; El Hariri et al., J. Pharm. Pharmacol.,1992, 44, 651-654).

Bile salts: The physiological role of bile includes the facilitation ofdispersion and absorption of lipids and fat-soluble vitamins (see e.g.,Malmsten, M. Surfactants and polymers in drug delivery, Informa HealthCare, New York, N.Y., 2002; Brunton, Chapter 38 in: Goodman & Gilman'sThe Pharmacological Basis of Therapeutics, 9th Ed., Hardman et al. Eds.,McGraw-Hill, New York, 1996, pp. 934-935). Various natural bile salts,and their synthetic derivatives, act as penetration enhancers. Thus theterm “bile salts” includes any of the naturally occurring components ofbile as well as any of their synthetic derivatives. Suitable bile saltsinclude, for example, cholic acid (or its pharmaceutically acceptablesodium salt, sodium cholate), dehydrocholic acid (sodiumdehydrocholate), deoxycholic acid (sodium deoxycholate), glucholic acid(sodium glucholate), glycholic acid (sodium glycocholate),glycodeoxycholic acid (sodium glycodeoxycholate), taurocholic acid(sodium taurocholate), taurodeoxycholic acid (sodium taurodeoxycholate),chenodeoxycholic acid (sodium chenodeoxycholate), ursodeoxycholic acid(UDCA), sodium tauro-24,25-dihydro-fusidate (STDHF), sodiumglycodihydrofusidate and polyoxyethylene-9-lauryl ether (POE) (see e.g.,Malmsten, M. Surfactants and polymers in drug delivery, Informa HealthCare, New York, N.Y., 2002; Lee et al., Critical Reviews in TherapeuticDrug Carrier Systems, 1991, page 92; Swinyard, Chapter 39 In:Remington's Pharmaceutical Sciences, 18th Ed., Gennaro, ed., MackPublishing Co., Easton, Pa., 1990, pages 782-783; Muranishi, CriticalReviews in Therapeutic Drug Carrier Systems, 1990, 7, 1-33; Yamamoto etal., J. Pharm. Exp. Ther., 1992, 263, 25; Yamashita et al., J. Pharm.Sci., 1990, 79, 579-583).

Chelating Agents: Chelating agents, as used in connection with thetechnology described herein, can be defined as compounds that removemetallic ions from solution by forming complexes therewith, with theresult that absorption of iRNAs through the mucosa is enhanced. Withregards to their use as penetration enhancers in the technologydescribed herein, chelating agents have the added advantage of alsoserving as DNase inhibitors, as most characterized DNA nucleases requirea divalent metal ion for catalysis and are thus inhibited by chelatingagents (Jarrett, J. Chromatogr., 1993, 618, 315-339). Suitable chelatingagents include but are not limited to disodiumethylenediaminetetraacetate (EDTA), citric acid, salicylates (e.g.,sodium salicylate, 5-methoxysalicylate and homovanilate), N-acylderivatives of collagen, laureth-9 and N-amino acyl derivatives ofbeta-diketones (enamines)(see e.g., Katdare, A. et al., Excipientdevelopment for pharmaceutical, biotechnology, and drug delivery, CRCPress, Danvers, Mass., 2006; Lee et al., Critical Reviews in TherapeuticDrug Carrier Systems, 1991, page 92; Muranishi, Critical Reviews inTherapeutic Drug Carrier Systems, 1990, 7, 1-33; Buur et al., J. ControlRel., 1990, 14, 43-51).

Non-chelating non-surfactants: As used herein, non-chelatingnon-surfactant penetration enhancing compounds can be defined ascompounds that demonstrate insignificant activity as chelating agents oras surfactants but that nonetheless enhance absorption of iRNAs throughthe alimentary mucosa (see e.g., Muranishi, Critical Reviews inTherapeutic Drug Carrier Systems, 1990, 7, 1-33). This class ofpenetration enhancers includes, for example, unsaturated cyclic ureas,1-alkyl- and 1-alkenylazacyclo-alkanone derivatives (Lee et al.,Critical Reviews in Therapeutic Drug Carrier Systems, 1991, page 92);and non-steroidal anti-inflammatory agents such as diclofenac sodium,indomethacin and phenylbutazone (Yamashita et al., J. Pharm. Pharmacol.,1987, 39, 621-626).

Agents that enhance uptake of iRNAs at the cellular level can also beadded to the pharmaceutical and other compositions of the technologydescribed herein. For example, cationic lipids, such as lipofectin(Junichi et al, U.S. Pat. No. 5,705,188), cationic glycerol derivatives,and polycationic molecules, such as polylysine (Lollo et al., PCTApplication WO 97/30731), are also known to enhance the cellular uptakeof dsRNAs. Examples of commercially available transfection reagentsinclude, for example Lipofectamine™ (Invitrogen; Carlsbad, Calif.),Lipofectamine2000™ (Invitrogen; Carlsbad, Calif.), 293Fectin™(Invitrogen; Carlsbad, Calif.), Cellfectin™ (Invitrogen; Carlsbad,Calif.), DMIRIE-C™ (Invitrogen; Carlsbad, Calif.), FreeStyle™ MAX(Invitrogen; Carlsbad, Calif.), Lipofectamine™ 2000 CD (Invitrogen;Carlsbad, Calif.), Lipofectamine™ (Invitrogen; Carlsbad, Calif.),RNAiMAX (Invitrogen; Carlsbad, Calif.), Oligofectamine™ (Invitrogen;Carlsbad, Calif.), Optifect™ (Invitrogen; Carlsbad, Calif.), X-tremeGENEQ2 Transfection Reagent (Roche; Grenzacherstrasse, Switzerland), DOTAPLiposomal Transfection Reagent (Grenzacherstrasse, Switzerland), DOSPERLiposomal Transfection Reagent (Grenzacherstrasse, Switzerland), orFugene (Grenzacherstrasse, Switzerland), Transfectam® Reagent (Promega;Madison, Wis.), TransFast™ Transfection Reagent (Promega; Madison,Wis.), Tfx™-20 Reagent (Promega; Madison, Wis.), Tfx™-50 Reagent(Promega; Madison, Wis.), DreamFect™ (OZ Biosciences; Marseille,France), EcoTransfect (OZ Biosciences; Marseille, France), TransPass^(a)D1 Transfection Reagent (New England Biolabs; Ipswich, Mass., USA),LyoVec™/LipoGen™ (Invitrogen; San Diego, Calif., USA), PerFectinTransfection Reagent (Genlantis; San Diego, Calif., USA), NeuroPORTERTransfection Reagent (Genlantis; San Diego, Calif., USA), GenePORTERTransfection reagent (Genlantis; San Diego, Calif., USA), GenePORTER 2Transfection reagent (Genlantis; San Diego, Calif., USA), CytofectinTransfection Reagent (Genlantis; San Diego, Calif., USA), BaculoPORTERTransfection Reagent (Genlantis; San Diego, Calif., USA), TroganPORTER™transfection Reagent (Genlantis; San Diego, Calif., USA), RiboFect(Bioline; Taunton, Mass., USA), PlasFect (Bioline; Taunton, Mass., USA),UniFECTOR (B-Bridge International; Mountain View, Calif., USA),SureFECTOR (B-Bridge International; Mountain View, Calif., USA), orHiFect™ (B-Bridge International, Mountain View, Calif., USA), amongothers.

Other agents can be utilized to enhance the penetration of theadministered nucleic acids, including glycols such as ethylene glycoland propylene glycol, pyrrols such as 2-pyrrol, azones, and terpenessuch as limonene and menthone.

Carriers

Certain compositions of the technology described herein also incorporatecarrier compounds in the formulation. As used herein, “carrier compound”or “carrier” can refer to a nucleic acid, or analog thereof, which isinert (i.e., does not possess biological activity per se) but isrecognized as a nucleic acid by in vivo processes that reduce thebioavailability of a nucleic acid having biological activity by, forexample, degrading the biologically active nucleic acid or promoting itsremoval from circulation. The coadministration of a nucleic acid and acarrier compound, typically with an excess of the latter substance, canresult in a substantial reduction of the amount of nucleic acidrecovered in the liver, kidney or other extracirculatory reservoirs,presumably due to competition between the carrier compound and thenucleic acid for a common receptor. For example, the recovery of apartially phosphorothioate dsRNA in hepatic tissue can be reduced whenit is coadministered with polyinosinic acid, dextran sulfate,polycytidic acid or 4-acetamido-4′isothiocyano-stilbene-2,2′-disulfonicacid (Miyao et al., DsRNA Res. Dev., 1995, 5, 115-121; Takakura et al.,DsRNA & Nucl. Acid Drug Dev., 1996, 6, 177-183.

Excipients

In contrast to a carrier compound, a “pharmaceutical carrier” or“excipient” is a pharmaceutically acceptable solvent, suspending agentor any other pharmacologically inert vehicle for delivering one or morenucleic acids to an animal. The excipient can be liquid or solid and isselected, with the planned manner of administration in mind, so as toprovide for the desired bulk, consistency, etc., when combined with anucleic acid and the other components of a given pharmaceuticalcomposition. Typical pharmaceutical carriers include, but are notlimited to, binding agents (e.g., pregelatinized maize starch,polyvinylpyrrolidone or hydroxypropyl methylcellulose, etc.); fillers(e.g., lactose and other sugars, microcrystalline cellulose, pectin,gelatin, calcium sulfate, ethyl cellulose, polyacrylates or calciumhydrogen phosphate, etc.); lubricants (e.g., magnesium stearate, talc,silica, colloidal silicon dioxide, stearic acid, metallic stearates,hydrogenated vegetable oils, corn starch, polyethylene glycols, sodiumbenzoate, sodium acetate, etc.); disintegrants (e.g., starch, sodiumstarch glycolate, etc.); and wetting agents (e.g., sodium laurylsulphate, etc).

Pharmaceutically acceptable organic or inorganic excipients suitable fornon-parenteral administration which do not deleteriously react withnucleic acids can also be used to formulate the compositions of thetechnology described herein. Suitable pharmaceutically acceptablecarriers include, but are not limited to, water, salt solutions,alcohols, polyethylene glycols, gelatin, lactose, amylose, magnesiumstearate, talc, silicic acid, viscous paraffin, hydroxymethylcellulose,polyvinylpyrrolidone and the like.

Formulations for topical administration of nucleic acids can includesterile and non-sterile aqueous solutions, non-aqueous solutions incommon solvents such as alcohols, or solutions of the nucleic acids inliquid or solid oil bases. The solutions can also contain buffers,diluents and other suitable additives. Pharmaceutically acceptableorganic or inorganic excipients suitable for non-parenteraladministration which do not deleteriously react with nucleic acids canbe used.

Suitable pharmaceutically acceptable excipients include, but are notlimited to, water, salt solutions, alcohol, polyethylene glycols,gelatin, lactose, amylose, magnesium stearate, talc, silicic acid,viscous paraffin, hydroxymethylcellulose, polyvinylpyrrolidone and thelike.

Other Components

The compositions of the technology described herein can additionallycontain other adjunct components conventionally found in pharmaceuticalcompositions, at their art-established usage levels. Thus, for example,the compositions can contain additional, compatible,pharmaceutically-active materials such as, for example, antipruritics,astringents, local anesthetics or anti-inflammatory agents, or cancontain additional materials useful in physically formulating variousdosage forms of the compositions of the technology described herein,such as dyes, flavoring agents, preservatives, antioxidants, opacifiers,thickening agents and stabilizers. However, such materials, when added,should not unduly interfere with the biological activities of thecomponents of the compositions of the technology described herein. Theformulations can be sterilized and, if desired, mixed with auxiliaryagents, e.g., lubricants, preservatives, stabilizers, wetting agents,emulsifiers, salts for influencing osmotic pressure, buffers, colorings,flavorings and/or aromatic substances and the like which do notdeleteriously interact with the nucleic acid(s) of the formulation.

Aqueous suspensions can contain substances which increase the viscosityof the suspension including, for example, sodium carboxymethylcellulose,sorbitol and/or dextran. The suspension can also contain stabilizers.

In some embodiments, pharmaceutical compositions featured in thetechnology described herein include (a) one or more iRNA compounds and(b) one or more agents which function by a non-RNAi mechanism and whichare useful in treating a liver disorder. Examples of such agentsinclude, but are not lmited to an anti-inflammatory agent,anti-steatosis agent, anti-viral, and/or anti-fibrosis agent. Inaddition, other substances commonly used to protect the liver, such assilymarin, can also be used in conjunction with the iRNAs describedherein. Other agents useful for treating liver diseases includetelbivudine, entecavir, and protease inhibitors such as telaprevir andother disclosed, for example, in Tung et al., U.S. ApplicationPublication Nos. 2005/0148548, 2004/0167116, and 2003/0144217; and inHale et al., U.S. Application Publication No. 2004/0127488.

Agents which can be useful in treating alpha-1 anti-trypsin liverdisorder include, but are not limited to, taurodoexycholic acid,selenium, PBA chaperone protein and/or compositions which increase theexpression of PBA chaperone protein, Prolastin®, and/or rAAV-AATadministration (Brantly et al., Hum Gene Ther 1177-86)

Agents useful in treating alcoholic liver disease include, but are notlimited to, oxandrolone and propylthiouracil.

Agents useful in treating chronic viral hepatitis include, but are notlimited to, alpha interferon, peginterferon, ribavirin, lamivudine, andadefovir dipivoxil.

Agents useful in treating autoimmune liver diseases include, but are notlimited to, prednisone and azathioprine.

Agents useful in treating steatorrhoeic hepatosis and non-alcoholicsteatohepatities include, but are not limited to, metformin andthiazolidinones such as pioglitazone, troglitizone, and rosiglitazone.

Agents useful in treating hepatic cancer include, but are not limitedto, chemotherapy and radiation. Accordingly, a treatment can include,for example, chemotherapy (for example, chlorambucil, prednisone,prednisolone, vincristine, cytarabine, clofarabine, farnesyl transferaseinhibitors, decitabine, inhibitors of MDR1), rituximab, interferon-α,anthracycline drugs (such as daunorubicin or idarubicin),L-asparaginase, doxorubicin, cyclophosphamide, doxorubicin, bleomycin,fludarabine, etoposide, pentostatin, or cladribine), bone marrowtransplant, stem cell transplant, anti-metabolite drugs (methotrexateand 6-mercaptopurine), or any combination thereof.

Chemotherapy is the treatment of cancer with drugs that can destroycancer cells. In current usage, the term “chemotherapy” usually refersto cytotoxic drugs which affect rapidly dividing cells in general, incontrast with targeted therapy. Chemotherapy drugs interfere with celldivision in various possible ways, e.g. with the duplication of DNA orthe separation of newly formed chromosomes. Most forms of chemotherapytarget all rapidly dividing cells and are not specific to cancer cells,although some degree of specificity can come from the inability of manycancer cells to repair DNA damage, while normal cells generally can.Most chemotherapy regimens are given in combination. Exemplarychemotherapeutic agents include, but are not limited to, 5-FU Enhancer,9-AC, AG2037, AG3340, Aggrecanase Inhibitor, Aminoglutethimide,Amsacrine (m-AMSA), Asparaginase, Azacitidine, Batimastat (BB94), BAY12-9566, BCH-4556, Bis-Naphtalimide, Busulfan, Capecitabine,Carboplatin, Carmustaine+Polifepr Osan, cdk4/cdk2 inhibitors,Chlorombucil, CI-994, Cisplatin, Cladribine, CS-682, Cytarabine HCl,D2163, Dactinomycin, Daunorubicin HCl, DepoCyt, Dexifosamide, Docetaxel,Dolastain, Doxifluridine, Doxorubicin, DX8951f, E 7070, EGFR,Epirubicin, Erythropoietin, Estramustine phosphate sodium, Etoposide(VP16-213), Farnesyl Transferase Inhibitor, FK 317, Flavopiridol,Floxuridine, Fludarabine, Fluorouracil (5-FU), Flutamide, Fragyline,Gemcitabine, Hexamethylmelamine (HMM), Hydroxyurea (hydroxycarbamide),Ifosfamide, Interferon Alfa-2a, Interferon Alfa-2b, Interleukin-2,Irinotecan, ISI 641, Krestin, Lemonal DP 2202, Leuprolide acetate(LHRH-releasing factor analogue), Levamisole, LiGLA (lithium-gammalinolenate), Lodine Seeds, Lometexol, Lomustine (CCNU), Marimistat,Mechlorethamine HCl (nitrogen mustard), Megestrol acetate, MeglamineGLA, Mercaptopurine, Mesna, Mitoguazone (methyl-GAG; methyl glyoxalbis-guanylhydrazone; MGBG), Mitotane (o.p′-DDD), Mitoxantrone,Mitoxantrone HCl, MMI 270, MMP, MTA/LY 231514, Octreotide, ODN 698,OK-432, Oral Platinum, Oral Taxoid, Paclitaxel (TAXOL®), PARPInhibitors, PD 183805, Pentostatin (2′ deoxycoformycin), PKC 412,Plicamycin, Procarbazine HCl, PSC 833, Ralitrexed, RAS FarnesylTransferase Inhibitor, RAS Oncogene Inhibitor, Semustine (methyl-CCNU),Streptozocin, Suramin, Tamoxifen citrate, Taxane Analog, Temozolomide,Teniposide (VM-26), Thioguanine, Thiotepa, Topotecan, Tyrosine Kinase,UFT (Tegafur/Uracil), Valrubicin, Vinblastine sulfate, Vindesinesulfate, VX-710, VX-853, YM 116, ZD 0101, ZD 0473/Anormed, ZD 1839, ZD9331

Toxicity and therapeutic efficacy of such compounds can be determined bystandard pharmaceutical procedures in cell cultures or experimentalanimals, e.g., for determining the LD50 (the dose lethal to 50% of thepopulation) and the ED50 (the dose therapeutically effective in 50% ofthe population). The dose ratio between toxic and therapeutic effects isthe therapeutic index and it can be expressed as the ratio LD50/ED50.Compounds that exhibit high therapeutic indices are preferred.

The data obtained from cell culture assays and animal studies can beused in formulating a range of dosage for use in humans. The dosage ofcompositions featured herein in the technology described herein liesgenerally within a range of circulating concentrations that include theED50 with little or no toxicity. The dosage can vary within this rangedepending upon the dosage form employed and the route of administrationutilized. For any compound used in the methods featured in thetechnology described herein, the therapeutically effective dose can beestimated initially from cell culture assays. A dose can be formulatedin animal models to achieve a circulating plasma concentration range ofthe compound or, when appropriate, of the polypeptide product of atarget sequence (e.g., achieving a decreased concentration of thepolypeptide) that includes the IC50 (i.e., the concentration of the testcompound which achieves a half-maximal inhibition of symptoms) asdetermined in cell culture. Such information can be used to moreaccurately determine useful doses in humans. Levels in plasma can bemeasured, for example, by high performance liquid chromatography.

In addition to their administration, as discussed above, the iRNAsfeatured in the technology described herein can be administered incombination with other known agents effective in treatment ofpathological processes mediated by Serpina1 expression. In any event,the administering physician can adjust the amount and timing of iRNAadministration on the basis of results observed using standard measuresof efficacy known in the art or described herein.

Methods for Treating or Preventing Diseases Caused by Expression of aSerpina1 Gene

The technology described herein relates to the use of an iRNA targetingSerpina1 and compositions containing at least one such iRNA for thetreatment or prevention of a Serpina1-mediated disorder or disease. Forexample, a composition containing an iRNA targeting a Serpina1 gene isused for treatment or prevention of liver disorders such as alpha-1anti-trypsin deficiency liver disease, chronic liver disease, liverinflammation, cirrhosis, liver fibrosis, and/or hepatocellularcarcinoma.

The technology described herein further relates to the use of an iRNA ora pharmaceutical composition thereof, e.g., for treating a liverdisorder, in combination with other pharmaceuticals and/or othertherapeutic methods, e.g., with known pharmaceuticals and/or knowntherapeutic methods, such as, for example, those which are currentlyemployed for treating these disorders. For example, in certainembodiments, an iRNA targeting Serpina1 is administered in combinationwith, e.g., an agent useful in treating a liver disorder as describedelsewhere herein.

The iRNA and an additional therapeutic agent can be administered in thesame combination, e.g., parenterally, or the additional therapeuticagent can be administered as part of a separate composition or byanother method known in the art or described herein.

The technology described herein features a method of administering aniRNA agent targeting Serpina1 to a patient having a disease or disordermediated by Serpina1 expression, such as a liver disorder.Administration of the dsRNA can resulting in a reduction of theseverity, signs, symptoms, and/or markers of such diseases or disordersin a patient with a liver disorder. By “reduction” in this context ismeant a statistically significant decrease in such level. The reductioncan be, for example, at least 10%, at least 20%, at least 30%, at least40% or more, and is preferably reduced to a level accepted as within therange of normal for an individual without such disorder.

Efficacy of treatment or prevention of disease can be assessed, forexample by measuring disease progression, disease remission, symptomseverity, reduction in pain, quality of life, dose of a medicationrequired to sustain a treatment effect, level of a disease marker or anyother measurable parameter appropriate for a given disease being treatedor targeted for prevention. It is well within the ability of one skilledin the art to monitor efficacy of treatment or prevention by measuringany one of such parameters, or any combination of parameters. Forexample, efficacy of treatment of liver fibrosis or amelioration ofliver fibrosis can be assessed, for example by periodic monitoring ofliver fibrosis markers: α-2-macroglobulin (a-MA), transferrin,apolipoproteinA1, hyaluronic acid (HA), laminin, N-terminal procollagenIII (PIIINP), 7S collagen IV (7S-IV), total bilirubin, indirectbilirubin, alanine aminotransferase (ALT), aspartate aminotransferase(AST), AST/ALT, g-glutamyl transpeptidase (GGT), alkaline phosphatase(ALP), albumin, albumin/globulin, blood urea nitrogen (BUN), creatinine(Cr), triglyceride, cholersterol, high density lipoprotein and lowdensity lipoprotein and liver puncture biopsy. Liver fibrosis markerscan be measured and/or liver puncture biopsy can be performed beforetreatment (initial readings) and subsequently (later readings) duringthe treatment regimen. Comparisons of the later readings with theinitial readings provide a physician an indication of whether thetreatment is effective. It is well within the ability of one skilled inthe art to monitor efficacy of treatment or prevention by measuring anyone of such parameters, or any combination of parameters. In connectionwith the administration of an iRNA targeting Serpina1 or pharmaceuticalcomposition thereof, “effective against” a hepatic fibrosis conditionindicates that administration in a clinically appropriate manner resultsin a beneficial effect for at least a statistically significant fractionof patients, such as a improvement of symptoms, a cure, a reduction indisease load, reduction in tumor mass or cell numbers, extension oflife, improvement in quality of life, or other effect generallyrecognized as positive by medical doctors familiar with treating liverfibrosis and the related causes.

The iRNA treatments described herein can be used to treat individualshaving the signs, symptoms and/or markers of, or being diagnosed with,or being a risk of having alpha-1 anti-trypsin deficiency liverdisorder, liver inflammation, cirrhosis, liver fibrosis, and/orhepatoceullar carcinoma. One of skill in the art can easily monitor thesigns, symptoms, and/or makers of such disorders in subjects receivingtreatment with iRNA as described herein and assay for a reduction inthese signs, symptoms and/or makers of at least 10% and preferably to aclinical level representing a low risk of liver disease.

A treatment or preventive effect is evident when there is astatistically significant improvement in one or more parameters ofdisease status, or by a failure to worsen or to develop symptoms wherethey would otherwise be anticipated. As an example, a favorable changeof at least 10% in a measurable parameter of disease, and preferably atleast 20%, 30%, 40%, 50% or more can be indicative of effectivetreatment. Efficacy for a given iRNA drug or formulation of that drugcan also be judged using an experimental animal model for the givendisease as known in the art. When using an experimental animal model,efficacy of treatment is evidenced when a statistically significantreduction in a marker or symptom is observed.

Alternatively, for certain indications, the efficacy can be measured byan increase in serum levels of Serpina1 protein. As an example, anincrease of serum levels of properly folded Serpina1 of at least 10%, atleast 20%, at least 50%, at least 100%, at least 200% more can beindicative of effective treatment.

Alternatively, the efficacy can be measured by a reduction in theseverity of disease as determined by one skilled in the art of diagnosisbased on a clinically accepted disease severity grading scale, as butone example the Child-Pugh score (sometimes the Child-Turcotte-Pughscore). In this example, prognosis of chronic liver disease, mainlycirrhosis, is measured by an aggregate score of five clinical measures,billirubin, serum albumin, INR, ascites, and hepatic encephalopathy.Each marker is assigned a value from 1-3, and the total value is used toprovide a score categorized as A (5-6 points), B (7-9 points), or C(10-15 points), which can be correlated with one and two year survivalrates. Methods for determination and analysis of Child-Pugh scores arewell known in the art (Farnsworth et al., Am J Surgery 2004 188:580-583;Child and Turcotte. Surgery and portal hypertension. In: The liver andportal hypertension. Edited by CG Child. Philadelphia: Saunders1964:50-64; Pugh et al., Br J Surg 1973; 60:648-52). Efficacy can bemeasured in this example by the movement of a patient from e.g., a “B”to an “A.” Any positive change resulting in e.g., lessening of severityof disease measured using the appropriate scale, represents adequatetreatment using an iRNA or iRNA formulation as described herein.

Patients can be administered a therapeutic amount of iRNA, such as 0.01mg/kg, 0.05 mg/kg, 0.1 mg/kg, 0.5 mg/kg, 1.0 mg/kg, 1.5 mg/kg, 2.0mg/kg, or 2.5 mg/kg dsRNA. The iRNA can be administered by intravenousinfusion over a period of time, such as over a 5 minute, 10 minute, 15minute, 20 minute, or 25 minute period. The administration is repeated,for example, on a regular basis, such as biweekly (i.e., every twoweeks) for one month, two months, three months, four months or longer.After an initial treatment regimen, the treatments can be administeredon a less frequent basis. For example, after administration biweekly forthree months, administration can be repeated once per month, for sixmonths or a year or longer. Administration of the iRNA can reduceSerpina1 levels, e.g., in a cell, tissue, blood, urine or othercompartment of the patient by at least 10%, at least 15%, at least 20%,at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, atleast 70%, at least 80% or at least 90% or more.

Before administration of a full dose of the iRNA, patients can beadministered a smaller dose, such as a 5% infusion reaction, andmonitored for adverse effects, such as an allergic reaction. In anotherexample, the patient can be monitored for unwanted immunostimulatoryeffects, such as increased cytokine (e.g., TNF-alpha or INF-alpha)levels.

Alpha-1 anti-trypsin deficiency liver disease can be hereditary.Therefore, a patient in need of a Serpina1 iRNA can be identified bytaking a family history. A healthcare provider, such as a doctor, nurse,or family member, can take a family history before prescribing oradministering a Serpina1 dsRNA. A DNA test can also be performed on thepatient to identify a mutation in the Serpina1 gene, before a Serpina1dsRNA is administered to the patient.

Owing to the inhibitory effects on Serpina1 expression, a compositionaccording to the technology described herein or a pharmaceuticalcomposition prepared therefrom can enhance the quality of life.

Methods for Inhibiting Expression of a Serpina1 Gene

In one aspect, provided herein is a method of inhibiting Serpina1expression in a cell, the method comprising: (a) introducing into thecell a dsRNA that targets a Serpina1 gene in the cell; and (b)maintaining the cell produced in step (a) for a time sufficient toobtain degradation of the mRNA transcript of a Serpina1 gene, therebyinhibiting expression of the Serpina1 gene in the cell. Reduction ingene expression can be assessed by any methods known it the art and bymethods, e. g. qRT-PCR, described herein.

In one embodiment, the cell is a mammalian cell, preferably a humancell. In another embodiment, the cell is a mammalian liver cell.

In one embodiment, the dsRNA is introduced to the cell, preferably, in aliposome, e.g. a LNP-formulated liposome known in the art and/ordescribed herein. In one embodiment, the LNP is formulated to target aspecific cell such as a hepatocyte.

In one embodiment, the Serpina1 expression is inhibited by at least,10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or even 100%.

In one aspect, provided herein is a method for inhibiting the expressionof a Serpina1gene in a mammal, the method comprising (a) administeringto the mammal a composition comprising a dsRNA that targets a Serpina1gene in a cell of the mammal; and (b) maintaining the mammal of step (a)for a time sufficient to obtain degradation of the mRNA transcript of aSerpina1 gene respectively, thereby inhibiting expression of theSerpina1 gene in the cell. Reduction in gene expression can be assessedby any methods known it the art and by methods, e. g. qRT-PCR, describedherein. In one embodiment, a puncture liver biopsy sample serves as thetissue material for monitoring the reduction in the target geneexpression.

In one embodiment, the method includes administering a compositionfeatured herein to the mammal such that expression of the targetSerpina1 gene is decreased, such as for an extended duration, e.g., atleast two, three, four days or more, e.g., one week, two weeks, threeweeks, or four weeks or longer.

Preferably, the iRNAs useful for the methods and compositions featuredherein specifically target RNAs (primary or processed) of the targetSerpina1gene. Compositions and methods for inhibiting the expression ofthese genes using iRNAs can be prepared and performed as describedelsewhere herein.

In one embodiment of the aspects described herein, the method includesadministering a composition containing an iRNA, where the iRNA includesa nucleotide sequence that is complementary to at least a part of an RNAtranscript of the Serpina1 gene of the mammal to be treated. When theorganism to be treated is a mammal such as a human, the composition canbe administered by any means known in the art including, but not limitedto oral, intraperitoneal, or parenteral routes, including intracranial(e.g., intraventricular, intraparenchymal and intrathecal), intravenous,intramuscular, subcutaneous, transdermal, airway (aerosol), nasal,rectal, and topical (including buccal and sublingual) administration. Incertain embodiments, the compositions are administered by intravenousinfusion or injection.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the iRNAs and methods featured in the invention,suitable methods and materials are described below. All publications,patent applications, patents, and other references mentioned herein areincorporated by reference in their entirety. In case of conflict, thepresent specification, including definitions, will control. In addition,the materials, methods, and examples are illustrative only and notintended to be limiting.

Some of the embodiments of the technology described herein can bedefined according to any of the following numbered paragraphs:

-   1. A double-stranded ribonucleic acid (dsRNA) for inhibiting    expression of Serpina1, wherein said dsRNA comprises a sense strand    and an antisense strand, wherein the sense strand comprises at least    15 contiguous nucleotides differing by no more than 3 nucleotides    from any of the nucleotide sequence of SEQ ID NO: 01 to SEQ ID NO:11    and the antisense strand comprises at least 15 contiguous    nucleotides differing by no more than 3 nucleotides from any of the    nucleotide sequence of SEQ ID NO: 14 to SEQ ID NO: 24.-   2. A double-stranded ribonucleic acid (dsRNA) for inhibiting    expression of Serpina1, wherein said dsRNA comprises a sense strand    and an antisense strand, the antisense strand comprising a region of    complementarity which comprises at least 15 contiguous nucleotides    differing by no more than 3 nucleotides from one of the antisense    sequences listed in Tables 3 and 4.-   3. The dsRNA of paragraph 2, wherein the sense and antisense strands    comprise sequences selected from the group composed of AD-44715.1,    AD-44722.1, AD-44734.1, AD-44717.1, AD-44723.1, AD-44735.1,    AD-44724.1, AD-44719.1, and AD-44737.1 of Table 3.-   4. The dsRNA of paragraph 1 or 2, wherein said dsRNA comprises at    least one modified nucleotide.-   5. The dsRNA of paragraph 4, wherein at least one of said modified    nucleotides is chosen from the group of: a 2′-O-methyl modified    nucleotide, a nucleotide comprising a 5′-phosphorothioate group, and    a terminal nucleotide linked to a cholesteryl derivative or    dodecanoic acid bisdecylamide group.-   6. The dsRNA of paragraph 4, wherein said modified nucleotide is    chosen from the group of: a 2′-deoxy-2′-fluoro modified nucleotide,    a 2′-deoxy-modified nucleotide, a locked nucleotide, an abasic    nucleotide, 2′-amino-modified nucleotide, 2′-alkyl-modified    nucleotide, morpholino nucleotide, a phosphoramidate, and a    non-natural base comprising nucleotide.-   7. The dsRNA of paragraph 2, wherein the region of complementarity    is at least 17 nucleotides in length.-   8. The dsRNA of paragraph 2, wherein the region of complementarity    is between 19 and 21 nucleotides in length.-   9. The dsRNA of paragraph 8, wherein the region of complementarity    is 19 nucleotides in length.-   10. The dsRNA of paragraph 1 or 2, wherein each strand is no more    than 30 nucleotides in length.-   11. The dsRNA of paragraph 1 or 2, wherein at least one strand    comprises a 3′ overhang of at least 1 nucleotide.-   12. The dsRNA of paragraph 1 or 2, wherein at least one strand    comprises a 3′ overhang of at least 2 nucleotides.-   13. The dsRNA of paragraph 1 or 2, further comprising a ligand.-   14 The dsRNA of paragraph 13, wherein the ligand is conjugated to    the 3′ end of the sense strand of the dsRNA.-   15. The dsRNA of paragraph 2, wherein the region of complementarity    consists of one of the antisense sequences of Tables 3 and 4.-   16. The dsRNA of paragraph 2, wherein the sense strand is consists    of any of SEQ ID NO: 01 to SEQ ID NO:11 and the antisense strand    consists of any of SEQ ID NO: 14 to SEQ ID NO: 24.-   17. The dsRNA of paragraph 1 or 2, wherein the dsRNA comprises a    sense strand consisting of a sense strand sequence selected from    Tables 3 and 4, and an antisense strand consisting of an antisense    sequence selected from Tables 3 and 4-   18. A cell containing the dsRNA of paragraph 1 or 2.-   19. A vector encoding at least one strand of a dsRNA, wherein said    dsRNA comprises a region of complementarity to at least a part of an    mRNA encoding Serpina1, wherein said dsRNA is 30 base pairs or less    in length, and wherein said dsRNA targets a said mRNA for cleavage.-   20. The vector of paragraph 19, wherein the region of    complementarity is at least 15 nucleotides in length.-   21. The vector of paragraph 19, wherein the region of    complementarity is 19 to 21 nucleotides in length.-   22. A cell comprising the vector of paragraph 19.-   23. A pharmaceutical composition for inhibiting expression of a    Serpina1 gene comprising the dsRNA of paragraph 1 or 2 or the vector    of paragraph 19.-   24. The pharmaceutical composition of paragraph 23, further    comprising a lipid formulation.-   25. The pharmaceutical composition of paragraph 23, wherein the    lipid formulation is a SNALP, or XTC formulation.-   26. A method of inhibiting Serpina1 expression in a cell, the method    comprising:    -   (a) introducing into the cell the dsRNA of paragraph 1 or 2 or        the vector of paragraph 19; and    -   (b) maintaining the cell produced in step (a) for a time        sufficient to obtain degradation of the mRNA transcript of a        Serpina1 gene, thereby inhibiting expression of the Serpina1        gene in the cell.-   27. The method of paragraph 26, wherein the Serpina1 expression is    inhibited by at least 30%.-   28. A method of treating a disorder mediated by Serpina1 expression    comprising administering to a patient in need of such treatment a    therapeutically effective amount of the dsRNA of paragraph 1 or 2 or    the vector of paragraph 19.-   29. The method of paragraph 28, wherein the disorder is Alpha 1    anti-trypsin deficiency liver disease.-   30. The method of paragraph 28, wherein the administration of the    dsRNA to the subject causes a decrease in cirrohsis, fibrosis,    and/or Serpina1 protein accumulation in the liver.-   31. The method of paragraph 28, wherein the dsRNA is administered at    a concentration of 0.01 mg/kg-5 mg/kg bodyweight of the patient.-   32. A method of reducing the likelihood of hepatocellular carcinoma    in a patient, wherein the method comprises administering to a    patient in need of such treatment a therapeutically effective amount    of the dsRNA of paragraph 1 or 2 or the vector of paragraph 19.-   33. The method of paragraph 32, wherein the likelihood of developing    hepatocellular carcinoma is reduced by a statistically significant    amount.-   34. The method of paragraph 32, wherein the dsRNA is administered at    a concentration of 0.01 mg/kg-5 mg/kg bodyweight of the patient.-   35. A method of reducing the accumulation of misfolded Serpina1    protein in the liver of a patient, wherein the method comprises    administering to a patient in need of such treatment a    therapeutically effective amount of the dsRNA of paragraph 1 or 2 or    the vector of paragraph 19.-   36. The method of paragraph 35, wherein the dsRNA is administered at    a concentration of 0.01 mg/kg-5 mg/kg bodyweight of the patient.-   37. A method of inhibiting the expression of Serpina1 in a patient,    wherein the method comprises administering to a patient in need of    such treatment a therapeutically effective amount of the dsRNA of    paragraph 1 or 2 or the vector of paragraph 19.-   38. The method of paragraph 37, wherein the dsRNA is administered at    a concentration of 0.01 mg/kg-5 mg/kg bodyweight of the patient.

EXAMPLES Example 1. IRNA Synthesis Source of Reagents

Where the source of a reagent is not specifically given herein, suchreagent can be obtained from any supplier of reagents for molecularbiology at a quality/purity standard for application in molecularbiology.

iRNA Design

The Serpina1 gene has multiple, alternate transcripts. siRNA design wascarried out to identify siRNAs targeting all human and rhesus (Macacamulatta) Serpina1 transcripts annotated in the NCBI Gene database(http://www.ncbi.nlm.nih.gov/gene/). Design used the followingtranscripts from the NCBI RefSeq collection: Human—NM_000295.4,NM_001002235.2, NM_001002236.2, NM_001127700.1, NM_001127701.1,NM_001127702.1, NM_001127703.1, NM_001127704.1, NM_001127705.1,NM_001127706.1, NM_001127707.1; Rhesus—XM_001098533.1, XM_001098837.1,XM_001098941.1, XM_001099044.1, XM_001099150.1, XM_0010992551 All siRNAduplexes were designed that shared 100% identity with all listed humanand rhesus transcripts.

Four hundred seventeen candidate siRNAs were used in a comprehensivesearch against the human transcriptome (defined as the set of NM_ andXM_ records within the human NCBI Refseq set). A total of 48 sense and48 antisense derived siRNA oligos were synthesized and formed intoduplexes.

iRNA Synthesis

Serpina1 tiled sequences were synthesized on MerMade 192 synthesizer at0.2 umol scale. Sequences that are mus specific and cross reactive inhum rhe and mus rat were synthesized. For all the sequences in the list,‘endolight’ chemistry was applied as detailed herein. All pyrimidines(cytosine and uridine) in the sense strand contained 2′-O-Methyl bases(2′ O-Methyl C and 2′-O-Methyl U). In the antisense strand, pyrimidinesadjacent to (towards 5′ position) ribo A nucleoside were replaced withtheir corresponding 2-O-Methyl nucleosides. A two base dTsdT extensionat 3′ end of both sense and anti sense sequences was then introduced.The sequence file was then converted to a text file to make itcompatible for loading in the MerMade 192 synthesis software

The synthesis of Serpina1 sequences used solid supported oligonucleotidesynthesis using phosphoramidite chemistry. The synthesis of thesequences described herein was performed at 1 um scale in 96 wellplates. The amidite solutions were prepared at 0.1M concentration andethyl thio tetrazole (0.6M in Acetonitrile) was used as activator.

The synthesized sequences were cleaved and deprotected in 96 wellplates, using methylamine in the first step and fluoride reagent in thesecond step. The crude sequences were precipitated using acetone:ethanol(80:20) mix and the pellet were re-suspended in 0.2M sodium acetatebuffer. Samples from each sequence were analyzed by LC-MS to confirm theidentity, UV for quantification and a selected set of samples by IEXchromatography to determine purity.

Serpina1 tiled sequences were precipitated and purified on AKTA Purifiersystem using Sephadex column. The process was run at ambienttemperature. Sample injection and collection was performed in 96 well(1.8 mL-deep well) plates. A single peak corresponding to the fulllength sequence was collected in the eluent. The desalted Serpina1sequences were analyzed for concentration (by UV measurement at A260)and purity (by ion exchange HPLC). The complementary single strands werethen combined in a 1:1 stoichiometric ratio to form siRNA duplexes. Adetailed list of Serpina1 single strands and duplexes are shown inTables 3 and 4.

Example 2. In Vitro Screening

HeLa or Hep3B cells (ATCC, Manassas, Va.) were grown to near confluenceat 37° C. in an atmosphere of 5% CO₂ in X (ATCC) supplemented with 10%FBS, streptomycin, and glutamine (ATCC) before being released from theplate by trypsinization. Transfection was carried out by adding 14.8 μlof Opti-MEM plus 0.2 μl of Lipofectamine RNAiMax per well (Invitrogen,Carlsbad Calif. cat #13778-150) to 5 μl of siRNA duplexes per well intoa 96-well plate and incubated at room temperature for 15 minutes. 80 μlof complete growth media without antibiotic containing ˜2×10⁴ HeLa orHep3B cells were then added to the siRNA mixture. Cells were incubatedfor either 24 or 120 hours prior to RNA purification. Single doseexperiments were performed at 10 nM and 0.1 nM final duplexconcentration and dose response experiments were done at 10, 1, 0.5,0.1, 0.05, 0.01, 0.005, 0.001, 0.0005, 0.0001, 0.00005, 0.00001 nM finalduplex concentration.

Total RNA was isolated using a Dynabeads® mRNA Isolation Kit(Invitrogen, part #610-12). Cells were harvested and lysed in 150 μl ofLysis/Binding Buffer then mixed for 5 minutes at 850 rpm using anEppendorf Thermomixer (the mixing speed was the same throughout theprocess). Ten microliters of magnetic beads and 80 μl Lysis/BindingBuffer mixture were added to a round bottom plate and mixed for 1minute. Magnetic beads were captured using a magnetic stand and thesupernatant was removed without disturbing the beads. After removingsupernatant, the lysed cells were added to the remaining beads and mixedfor 5 minutes. After removing supernatant, magnetic beads were washed 2times with 150 μl Wash Buffer A and mixed for 1 minute. Beads werecaptured again and supernatant removed. Beads were then washed with 150μl Wash Buffer B, captured and supernatant was removed. Beads were nextwashed with 150 μl Elution Buffer, captured and supernatant removed.Beads were allowed to dry for 2 minutes. After drying, 50 μl of ElutionBuffer was added and mixed for 5 minutes at 70° C. Beads were capturedon magnet for 5 minutes. 40 μl of supernatant was removed and added toanother 96 well plate.

cDNA synthesis was performed using ABI High capacity cDNA reversetranscription kit (Applied Biosystems, Foster City, Calif., Cat#4368813). A master mix of 2 μl 10× Buffer, 0.8 μl 25×dNTPs, 2 μl Randomprimers, 1 μl Reverse Transcriptase, 1 μl RNase inhibitor and 3.41 ofH2O per reaction were added into 10 μtotal RNA. cDNA was generated usinga Bio-Rad C-1000 or S-1000 thermal cycler (Hercules, Calif.) through thefollowing steps: 25° C. 10 min, 37° C. 120 min, 85° C. 5 sec, 4° C.hold.

To perform real-time PCR, 2 μl of cDNA were added to a master mixcontaining 0.5 μl GAPDH TaqMan Probe (Applied Biosystems Cat #4326317E),0.5 μl Serpina1 TaqMan probe (Applied Biosystems cat # Hs00165475_m1)and 5 μl Lightcycler 480 probe master mix (Roche Cat #04887301001) perwell in a 384 well 50 plates (Roche cat #04887301001). Real time PCR wasdone in an ABI 7900HT Real Time PCR system (Applied Biosystems) usingthe ΔΔCt(RQ) assay. Each duplex was tested in two independenttransfections and each transfection was assayed in duplicate, unlessotherwise noted in the summary tables.

To calculate relative fold change, real time data were analyzed usingthe ΔΔCt method and normalized to assays performed with cellstransfected with 10 nM AD-1955, or mock transfected cells (FIG. 1 andTable 5). IC50s were calculated using a 4 parameter fit model usingXLFit and normalized to cells transfected with AD-1955 over the samedose range, or to its own lowest dose (Tables 6 and 7). In Table 7, tworepetitions are shown.

To determine the effect of the iRNAs described herein on expression ofmutant Serpina1 alleles, the inhibitory effect of AD-44715 on the PiZallele was determined. The wild-type allele of Serpina1 (WT-hAAT) wasmutated to incorporate the E443K mutation that distinguishes the PiZallele from the wildtype allele. 293T cells were transfected withplasmids expressing wildtype or PiZ Serpina1. After 4 hours, cells weretransfected with AD-44715. Twenty-four hours later, RNA was isolated andquantitative PCR performed as described herein. Data were normalized tothe expression level of hGAPDH in the 293T cells and are presented inarbitrary units (FIG. 2). The results indicate that the PiZ allele issubject to silencing by AD-44715.

Example 3. In Vivo Tests

In vivo tests of the AD-44715 were then performed. CD1 mice received a 3mL hydrodynamic intravenous injection of PBS or a plamid expressingeither myc-tagged wildtype Serpina1 (WT_mycAAT) or a myc-tagged PiZallele of Serpina1 (Z-mycAAT) on day 1. On day 4, the mice were given anintravenous injection of PBS, or the AF11 LNP formulation of AD-44715 orAD-1955 iRNAs at 1 mg/kg (the AF11 LNP formulation was used throughoutthese animal studies). On day 6 the mice were sacrificed and expressionof Serpina1 mRNA was determined as described herein. FIG. 3 shows theexpression of human Serpina1 in arbitrary units, normalized to mouseGAPDH. The results indicate that the AD-44715 duplex inhibits expressionof both wildtype and mutant Serpina1 transcripts in vivo.

Transgenic mice expressing the Z-AAT form of human AAT were used toexamine the effect of the iRNAs. The level of serum AAT mRNA and proteinwas measured 48 hours after intravenous administration of AD-44715 atdoses of 1, 0.3, 0.1 and 0.03 mg/kg and AD-1955 at 1 mg/kg (FIG. 4(mRNA); FIG. 5 (protein)). The siRNAs were administered as AF11 LNPformulations as described herein. Levels of serum AAT were measured onDay 0 and at 48 hours after administration, following sacrifice of theanimals. A decrease in both mRNA and protein levels of AAT is observedat 48 hours in a dose-dependent manner following administration ofAD-44715.

Levels of AAT protein in the liver of the animals were also determinedat 48 hours (FIGS. 6A-6B). FIGS. 6A-6B show the level of AAT monomerpresent in the liver samples. The monomer is obtained from the solublefraction of liver homogenate and a decrease in monomer levels isobserved at 48 hours in a dose-dependent manner following administrationof an AF11 LNP formulation of AD-44715. FIGS. 7A-7B show the level ofAAT polymer present in the liver samples. No change in polymer AATlevels was observed in this single, short-term timepoint experiment.FIGS. 8A-8C show monomer and polymer forms of AAT at various dosages ofAF11 LNP formulations of AD-44715 (FIGS. 8A and 8B, respectively) andthe ratio of monomer to polymer AAT (FIG. 8C) in the liver samples. TheED₅₀ in mice was 0.03-0.1 mg/kg.

A second iRNA demonstrated similar results when administered to femaletransgenic mice expressing the Z-AAT form of human AAT. The levels ofAAT protein in the serum and the mRNA levels of AAT in the livers of themice were measured 48 hours after intravenous administration of PBS oran AF11 LNP formulation of AD-44724 at a dose of 0.3 mg/kg or AD-1955luciferase control at 0.3 mg/kg (FIGS. 9A-9B). Levels of serum AAT weremeasured on Day 0 and at 48 hours after administration, followingsacrifice of the animals. Each experimental group contained 3 animals. Adecrease of more than 90% in both serum and liver levels of AAT wasobserved at 48 hours following administration of AD-44724.

A multi-dose experiment was conducted in male transgenic mice expressingthe Z-AAT form of human AAT. Mice were given 3 weekly doses of iRNA asan AF11 LNP formulation; either AD-44715 or AD-1955 luciferase controlat 0.3 mg/kg (FIG. 10A). Each experimental group consisted of fiveanimals. Serum levels of AAT protein were measured at the time of eachdose of iRNA. Forty-eight hours after the third dose was administered,animals were sacrificed and serum protein and liver mRNA levels of AATwere measured (FIGS. 10B-10C). AD-44715 decreased AAT levels at alltimepoints after administration. The decrease of AAT mRNA in both theliver and serum at the time of sacrifice was approximately 90%.

Another multi-dose experiment was conducted in male transgenic miceexpressing the Z-AAT form of human AAT to investigate the effect oflonger term dosage on momomer and polymer forms of AAT in the liver.Mice were given 3 weekly doses of either AD44715 AAT iRNA or AD1955luciferase control iRNA at 0.3 mg/kg as AF11 LNP formulations (FIG.11A). Each experimental group consisted of five animals. Forty-eighthours after the third dose was administered, animals were sacrificed andmonomeric and polymeric levels of AAT protein in the liver were measuredby Western blot. FIGS. 11B and 11C show a summary graph and Westernblot, respectively, showing monomeric AAT levels in the livers ofanimals treated with siLuc control and AAT iRNA at the time ofsacrifice. Administration of AD-44715 decreased AAT monomers byapproximately 90%.

FIGS. 11D and 11E show a summary graph and Western blot respectively forpolymeric AAT levels in the animals treated according to the regimen inFIG. 11A. AAT polymer in the livers was decreased at the time ofsacrifice by approximately 20%.

In order to examine the effects of longer-term AAT inhibition, liversections obtained from mice treated according to the dosing scheme shownin FIG. 11A were stained with periodic acid-Schiff stain, in whichpolymer globules appear pink. Sections from tissue obtained 48 hoursafter the last dose were examined. When viewed at 200× magnification,much less pink stain was visible in sections from animals receiving theAAT-specific (AD-44715) iRNA as compared to sections from animals in thecontrol group (siLUC; AD-1955).

A duration study was conducted, in which mice received a single dose ofAAT-specific (AD-44715) or control siRNA (Factor VII siRNA) as AF11 LNPformulations at a dosage of 0.3 mg/kg and samples were analyzed through14 days post treatment. AAT levels were measured as serum protein (FIG.12A) and mRNA in the liver (FIG. 12B). The level of AAT decreased bymore than 95% on Day 2 and began to rise thereafter. On Day 4, AAT wasdecreased approximately 90%, on Day 7 30-50%, and by Day 10 was atnormal levels. FIG. 12C shows the level of knockdown of Factor VII. Thecontrol doses of Factor VII-specific siRNA reduced Factor VII expressionby the percentage expected at the administered dose.

Example 4: Extended Dosing Regimes

AAT-specific iRNAs can be administered at 0.3 mg/kg over the course ofseveral weeks, including dosing every other week for a total of 6 doses.The effect of iRNA administration is assayed by weekly serum bleeds tomeasure serum mRNA, protein levels of AAT and conduct liver functiontests. Additional analysis for effects on liver function can include,for example, analysis of liver tissue to measure AAT mRNA, AAT protein,liver monomer, and liver polymer at the time of sacrifice; staining ofliver tissue with PAS to measure globules or staining with Sirius red orH&E to examine liver histology at the time of sacrifice; and the use ofBrdU incorporation to examine cell proliferation.

Example 5: Administration of siRNAs to Subjects with Liver Disease

AAT-specific siRNAs can be administered to mice displaying symptoms ofalpha-1 anti-trypsin related liver disease. The effect of siRNAs on cellproliferation and/or cell division in these mice is examined bystaining, for example, for Ki-67, PCNA, or BrdU incorporation. The doseresponse of a diseased liver to AAT-specific siRNA is measured byassaying the expression of AAT protein and/or mRNA or using anyparameter of liver function known in the art, including liver functiontests or histological examination as described above herein.

Example 6: Long Term Dosing

AAT-specific siRNAs were administered to mice displaying symptoms ofalpha-1 anti-trypsin related liver disease for extended dosing regimes.The experimental design is depicted in FIG. 13A. Transgenic male miceexpressing Z-AAT were administered 0.3 mg/kg doses of siRNA (eitherLNP-AAT or control LNP-Luc; N=6 for each group) every other week for atotal of 7 doses. The experiment was conducted with human AAT-specificreagents. On day 86 the mice were sacrificed. Liver and serum sampleswere collected and liver samples were subjected to mRNA, protein, PASstain, and BrdU analysis.

Analysis of human AAT mRNA levels in liver samples demonstrated that theLNP-AAT treated mice displayed lower levels of AAT expression (FIG.13B). Analysis of human AAT protein levels likewise indicated reducedlevels of both monomer and polymer forms of AAT in LNP-AAT treated miceas compared to controls (FIGS. 14A-14C).

Cell division is a means by which the liver replaces injuredhepatocytes. BrdU pumps were implanted in 3 mice from each treatmentgroup at day 83 and BrdU incorporation in the liver samples of theseanimals was determined. The LNP-AAT treated mice demonstrated lowerlevels of BrdU incorporation, indicating that treatment with LNP-AAT wasbeneficial to the health of the liver (FIG. 15).

Collagen levels in the liver of the mice were also examined bydetermining mRNA levels. Col1A1, Col1a2 (data not shown) and Col3a1(FIG. 16) were expressed at lower levels in the livers of LNP-AATtreated mice as compared to control LNP-Luc treated mice and wascomparable to the level of expression in wild-type parent animals.Decreased collagen expression is indicative of reduced fibrosis.

Liver cells of LNP-Luc treated mice and LNP-AAT treated mice were alsoexamined by electron microscopy (FIG. 17). Animals treated with LNP-AAThad cells with smaller and fewer globules, less ER dilation, fewerautophagic vacuoles, and less mitochondrial injury. Liver ultrastructurewas also observed to be markedly improved after LNP-AAT treatment.

This long term dosing experiment demonstrates that LNP-AAT dosing onalternate weeks, at low dosage, is very effective in decreasing diseasephenotypes. In LNP-AAT treated mice, levels of AAT mRNA and protein inthe liver were decreased, PAS stain (data not shown) decreased, liverdamage, as measured by BrdU incorporation was lower, and fibrosis wasreduced.

TABLE 2 Abbreviations of nucleotide monomers used in nucleic acidsequence representation. It will be understood that these monomers, whenpresent in an oligonucleotide, are mutually linked by5′-3′-phosphodiester bonds. Abbreviation Nucleotide(s) A adenosine Ccytidine G guanosine T thymidine U uridine N any nucleotide (G, A, C, Tor U) a 2′-O-methyladenosine c 2′-O-methylcytidine g2′-O-methylguanosine u 2′-O-methyluridine dT 2′-deoxythymidine sphosphorothioate linkage

TABLE 3Unmodified Sense and antisense strand sequences of Serpinal dsRNAsSense sequence Antisensesequence (SEQ ID NOS 29- (SEQ ID NOS 73-116,Position 72, respectively, in  Antisense respectively, in  relative toDuplex ID Sense ID order of appearance) ID order of appearance)NM_000295.4 AD-44697.1 A-93465.1 CUGGCACACCAGUCCAACA A-93466.1UGUUGGACUGGUGUGCCAG 454-472 AD-44703.1 A-93467.1 AGUCCAACAGCACCAAUAUA-93468.1 AUAUUGGUGCUGUUGGACU 464-482 AD-44709.1 A-93469.1UCCAACAGCACCAAUAUCU A-93470.1 AGAUAUUGGUGCUGUUGGA 466-484 AD-44715.1A-93471.1 CCAACAGCACCAAUAUCUU A-93472.1 AAGAUAUUGGUGCUGUUGG 467-485AD-44721.1 A-93473.1 AACAGCACCAAUAUCUUCU A-93474.1 AGAAGAUAUUGGUGCUGUU469-487 AD-44727.1 A-93475.1 CUCCCCAGUGAGCAUCGCU A-93476.1AGCGAUGCUCACUGGGGAG 489-507 AD-44733.1 A-93477.1 CCCAGUGAGCAUCGCUACAA-93478.1 UGUAGCGAUGCUCACUGGG 492-510 AD-44692.1 A-93479.1GUGAGCAUCGCUACAGCCU A-93480.1 AGGCUGUAGCGAUGCUCAC 496-514 AD-44698.1A-93481.1 UGAGCAUCGCUACAGCCUU A-93482.1 AAGGCUGUAGCGAUGCUCA 497-515AD-44704.1 A-93483.1 GAGCAUCGCUACAGCCUUU A-93484.1 AAAGGCUGUAGCGAUGCUC498-516 AD-44710.1 A-93485.1 CAUCGCUACAGCCUUUGCA A-93486.1UGCAAAGGCUGUAGCGAUG 501-519 AD-44716.1 A-93487.1 CUACAGCCUUUGCAAUGCUA-93488.1 AGCAUUGCAAAGGCUGUAG 506-524 AD-44722.1 A-93489.1GGACCAAGGCUGACACUCA A-93490.1 UGAGUGUCAGCCUUGGUCC 533-551 AD-44728.1A-93491.1 AUCCUGGAGGGCCUGAAUU A-93492.1 AAUUCAGGCCCUCCAGGAU 559-577AD-44734.1 A-93493.1 UCCUGGAGGGCCUGAAUUU A-93494.1 AAAUUCAGGCCCUCCAGGA560-578 AD-44699.1 A-93497.1 UGGAUAAGUUUUUGGAGGA A-93498.1UCCUCCAAAAACUUAUCCA 713-731 AD-44705.1 A-93499.1 GGAUAAGUUUUUGGAGGAUA-93500.1 AUCCUCCAAAAACUUAUCC 714-732 AD-44711.1 A-93501.1GACACCGAAGAGGCCAAGA A-93502.1 UCUUGGCCUCUUCGGUGUC 778-796 AD-44717.1A-93503.1 ACACCGAAGAGGCCAAGAA A-93504.1 UUCUUGGCCUCUUCGGUGU 779-797AD-44723.1 A-93505.1 CACCGAAGAGGCCAAGAAA A-93506.1 UUUCUUGGCCUCUUCGGUG780-798 AD-44729.1 A-93507.1 AAGAGGCCAAGAAACAGAU A-93508.1AUCUGUUUCUUGGCCUCUU 785-803 AD-44735.1 A-93509.1 GAGGCCAAGAAACAGAUCAA-93510.1 UGAUCUGUUUCUUGGCCUC 787-805 AD-44694.1 A-93511.1AUUUGGUCAAGGAGCUUGA A-93512.1 UCAAGCUCCUUGACCAAAU 845-863 AD-44700.1A-93513.1 GGUCAAGGAGCUUGACAGA A-93514.1 UCUGUCAAGCUCCUUGACC 849-867AD-44706.1 A-93515.1 GGAGCUUGACAGAGACACA A-93516.1 UGUGUCUCUGUCAAGCUCC855-873 AD-44712.1 A-93517.1 AGCUUGACAGAGACACAGU A-93518.1ACUGUGUCUCUGUCAAGCU 857-875 AD-44718.1 A-93519.1 ACAGUUUUUGCUCUGGUGAA-93520.1 UCACCAGAGCAAAAACUGU 871-889 AD-44724.1 A-93521.1CAGUUUUUGCUCUGGUGAA A-93522.1 UUCACCAGAGCAAAAACUG 872-890 AD-44730.1A-93523.1 UUUGCUCUGGUGAAUUACA A-93524.1 UGUAAUUCACCAGAGCAAA 877-895AD-44736.1 A-93525.1 UUGCUCUGGUGAAUUACAU A-93526.1 AUGUAAUUCACCAGAGCAA878-896 AD-44695.1 A-93527.1 UACAUCUUCUUUAAAGGCA A-93528.1UGCCUUUAAAGAAGAUGUA 892-910 AD-44701.1 A-93529.1 AAUGGGAGAGACCCUUUGAA-93530.1 UCAAAGGGUCUCUCCCAUU 911-929 AD-44707.1 A-93531.1AGGAAGAGGACUUCCACGU A-93532.1 ACGUGGAAGUCCUCUUCCU 944-962 AD-44713.1A-93533.1 CGUUUAGGCAUGUUUAACA A-93534.1 UGUUAAACAUGCCUAAACG 1000-1018AD-44719.1 A-93535.1 GUUUAGGCAUGUUUAACAU A-93536.1 AUGUUAAACAUGCCUAAAC1001-1019 AD-44725.1 A-93537.1 UGGGUGCUGCUGAUGAAAU A-93538.1AUUUCAUCAGCAGCACCCA 1045-1063 AD-44731.1 A-93539.1 UUCCUGCCUGAUGAGGGGAA-93540.1 UCCCCUCAUCAGGCAGGAA 1090-1108 AD-44737.1 A-93541.1CCUGCCUGAUGAGGGGAAA A-93542.1 UUUCCCCUCAUCAGGCAGG 1092-1110 AD-44696.1A-93543.1 AGCACCUGGAAAAUGAACU A-93544.1 AGUUCAUUUUCCAGGUGCU 1115-1133AD-44702.1 A-93545.1 UGGAAAAUGAACUCACCCA A-93546.1 UGGGUGAGUUCAUUUUCCA1121-1139 AD-44714.1 A-93549.1 CCAUUACUGGAACCUAUGA A-93550.1UCAUAGGUUCCAGUAAUGG 1208-1226 AD-44720.1 A-93551.1 CAUUACUGGAACCUAUGAUA-93552.1 AUCAUAGGUUCCAGUAAUG 1209-1227 AD-44726.1 A-93553.1UUACUGGAACCUAUGAUCU A-93554.1 AGAUCAUAGGUUCCAGUAA 1211-1229 AD-44732.1A-93555.1 ACUGGAACCUAUGAUCUGA A-93556.1 UCAGAUCAUAGGUUCCAGU 1213-1231

TABLE 4 Modified Sense and antisense strand sequences of Serpinal dsRNAsSense strand sequence Antisense sequence (SEQ ID NOS 117-160,(SEQ ID NOS 161-204, Duplex Sense respectively, in order Antisenserespectively, in order ID strand ID of appearance) ID of appearance)AD-44697.1 A-93465.1 cuGGcAcAccAGuccAAcAdTsdT A-93466.1UGUUGGACUGGUGUGCcAGdTsdT AD-44703.1 A-93467.1 AGuccAAcAGcAccAAuAudTsdTA-93468.1 AuAUUGGUGCUGUUGGACUdTsdT AD-44709.1 A-93469.1uccAAcAGcAccAAuAucudTsdT A-93470.1 AGAuAUUGGUGCUGUUGGAdTsdT AD-44715.1A-93471.1 ccAAcAGcAccAAuAucuudTsdT A-93472.1 AAGAuAUUGGUGCUGUUGGdTsdTAD-44721.1 A-93473.1 AAcAGcAccAAuAucuucudTsdT A-93474.1AGAAGAuAUUGGUGCUGUUdTsdT AD-44727.1 A-93475.1 cuccccAGuGAGcAucGcudTsdTA-93476.1 AGCGAUGCUcACUGGGGAGdTsdT AD-44733.1 A-93477.1cccAGuGAGcAucGcuAcAdTsdT A-93478.1 UGuAGCGAUGCUcACUGGGdTsdT AD-44692.1A-93479.1 GuGAGcAucGcuAcAGccudTsdT A-93480.1 AGGCUGuAGCGAUGCUcACdTsdTAD-44698.1 A-93481.1 uGAGcAucGcuAcAGccuudTsdT A-93482.1AAGGCUGuAGCGAUGCUcAdTsdT AD-44704.1 A-93483.1 GAGcAucGcuAcAGccuuudTsdTA-93484.1 AAAGGCUGuAGCGAUGCUCdTsdT AD-44710.1 A-93485.1cAucGcuAcAGccuuuGcAdTsdT A-93486.1 UGcAAAGGCUGuAGCGAUGdTsdT AD-44716.1A-93487.1 cuAcAGccuuuGcAAuGcudTsdT A-93488.1 AGcAUUGcAAAGGCUGuAGdTsdTAD-44722.1 A-93489.1 GGAccAAGGcuGAcAcucAdTsdT A-93490.1UGAGUGUcAGCCUUGGUCCdTsdT AD-44728.1 A-93491.1 AuccuGGAGGGccuGAAuudTsdTA-93492.1 AAUUcAGGCCCUCcAGGAUdTsdT AD-44734.1 A-93493.1uccuGGAGGGccuGAAuuudTsdT A-93494.1 AAAUUcAGGCCCUCcAGGAdTsdT AD-44699.1A-93497.1 uGGAuAAGuuuuuGGAGGAdTsdT A-93498.1 UCCUCcAAAAACUuAUCcAdTsdTAD-44705.1 A-93499.1 GGAuAAGuuuuuGGAGGAudTsdT A-93500.1AUCCUCcAAAAACUuAUCCdTsdT AD-44711.1 A-93501.1 GAcAccGAAGAGGccAAGAdTsdA-93502.1 UCUUGGCCUCUUCGGUGUCdTsdT AD-44717.1 A-93503.1AcAccGAAGAGGccAAGAAdTsd A-93504.1 UUCUUGGCCUCUUCGGUGUdTsdT AD-44723.1A-93505.1 cAccGAAGAGGccAAGAAAdTsd A-93506.1 UUUCUUGGCCUCUUCGGUGdTsdTAD-44729.1 A-93507.1 AAGAGGccAAGAAAcAGAudTsd A-93508.1AUCUGUUUCUUGGCCUCUUdTsdT AD-44735.1 A-93509.1 GAGGccAAGAAAcAGAucAdTsdA-93510.1 UGAUCUGUUUCUUGGCCUCdTsdT AD-44694.1 A-93511.1AuuuGGucAAGGAGcuuGAdTsdT A-93512.1 UcAAGCUCCUUGACcAAAUdTsdT AD-44700.1A-93513.1 GGucAAGGAGcuuGAcAGAdTsdT A-93514.1 UCUGUcAAGCUCCUUGACCdTsdTAD-44706.1 A-93515.1 GGAGcuuGAcAGAGAcAcAdTsdT A-93516.1UGUGUCUCUGUcAAGCUCCdTsdT AD-44712.1 A-93517.1 AGcuuGAcAGAGAcAcAGudTsdTA-93518.1 ACUGUGUCUCUGUcAAGCUdTsdT AD-44718.1 A-93519.1AcAGuuuuuGcucuGGuGAdTsdT A-93520.1 UcACcAGAGcAAAAACUGUdTsdT AD-44724.1A-93521.1 cAGuuuuuGcucuGGuGAAdTsdT A-93522.1 UUcACcAGAGcAAAAACUGdTsdTAD-44730.1 A-93523.1 uuuGcucuGGuGAAuuAcAdTsdT A-93524.1UGuAAUUcACcAGAGcAAAdTsdT AD-44736.1 A-93525.1 uuGcucuGGuGAAuuAcAudTsdTA-93526.1 AUGuAAUUcACcAGAGcAAdTsdT AD-44695.1 A-93527.1uAcAucuucuuuAAAGGcAdTsdT A-93528.1 UGCCUUuAAAGAAGAUGuAdTsdT AD-44701.1A-93529.1 AAuGGGAGAGAcccuuuGAdTsdT A-93530.1 UcAAAGGGUCUCUCCcAUUdTsdTAD-44707.1 A-93531.1 AGGAAGAGGAcuuccAcGudTsdT A-93532.1ACGUGGAAGUCCUCUUCCUdTsdT AD-44713.1 A-93533.1 cGuuuAGGcAuGuuuAAcAdTsdTA-93534.1 UGUuAAAcAUGCCuAAACGdTsdT AD-44719.1 A-93535.1GuuuAGGcAuGuuuAAcAudTsdT A-93536.1 AUGUuAAAcAUGCCuAAACdTsdT AD-44725.1A-93537.1 uGGGuGcuGcuGAuGAAAudTsdT A-93538.1 AUUUcAUcAGcAGcACCcAdTsdTAD-44731.1 A-93539.1 uuccuGccuGAuGAGGGGAdTsdT A-93540.1UCCCCUcAUcAGGcAGGAAdTsdT AD-44737.1 A-93541.1 ccuGccuGAuGAGGGGAAAdTsdTA-93542.1 UUUCCCCUcAUcAGGcAGGdTsdT AD-44696.1 A-93543.1AGcAccuGGAAAAuGAAcudTsdT A-93544.1 AGUUcAUUUUCcAGGUGCUdTsdT AD-44702.1A-93545.1 uGGAAAAuGAAcucAcccAdTsdT A-93546.1 UGGGUGAGUUcAUUUUCcAdTsdTAD-44714.1 A-93549.1 ccAuuAcuGGAAccuAuGAdTsdT A-93550.1UcAuAGGUUCcAGuAAUGGdTsdT AD-44720.1 A-93551.1 cAuuAcuGGAAccuAuGAudTsdTA-93552.1 AUcAuAGGUUCcAGuAAUGdTsdT AD-44726.1 A-93553.1uuAcuGGAAccuAuGAucudTsdT A-93554.1 AGAUcAuAGGUUCcAGuAAdTsdT AD-44732.1A-93555.1 AcuGGAAccuAuGAucuGAdTsdT A-93556.1 UcAGAUcAuAGGUUCcAGUdTsdT

TABLE 5 Serpina1 single dose screen 10 nM Average 0.1 nM AverageAD-44692.1 1.09 1.03 AD-44694.1 0.40 1.01 AD-44695.1 0.67 1.12AD-44696.1 0.73 1.11 AD-44697.1 0.37 1.08 AD-44697.1 0.29 1.13AD-44698.1 0.35 0.97 AD-44699.1 0.51 1.41 AD-44700.1 0.15 0.90AD-44701.1 0.58 1.22 AD-44702.1 0.45 0.84 AD-44703.1 0.16 0.99AD-44703.1 0.13 1.05 AD-44704.1 0.82 1.22 AD-44705.1 0.10 0.95AD-44706.1 0.31 1.07 AD-44707.1 0.21 1.13 AD-44709.1 0.09 0.92AD-44709.1 0.12 0.99 AD-44710.1 1.09 0.92 AD-44711.1 0.14 1.05AD-44712.1 0.72 1.26 AD-44713.1 0.10 1.04 AD-44714.1 0.72 1.34AD-44715.1 0.02 0.18 AD-44715.1 0.03 0.23 AD-44716.1 0.99 1.08AD-44717.1 0.04 0.52 AD-44718.1 0.76 1.14 AD-44719.1 0.03 0.53AD-44720.1 0.36 1.14 AD-44721.1 0.91 1.12 AD-44722.1 0.03 0.40AD-44723.1 0.09 0.70 AD-44724.1 0.02 0.37 AD-44725.1 0.65 1.29AD-44726.1 0.44 1.11 AD-44727.1 1.15 1.27 AD-44728.1 0.67 1.24AD-44729.1 0.52 0.97 AD-44730.1 0.58 1.11 AD-44731.1 0.93 1.26AD-44732.1 0.16 0.94 AD-44733.1 0.10 0.85 AD-44734.1 0.03 0.52AD-44735.1 0.04 0.48 AD-44736.1 0.12 0.99 AD-44737.1 0.25 0.94 Mock 1.171.10 Mock 0.87 1.15 AD-1955 1.12 0.87 AD-1955 1.01 0.93 AD-1955 0.861.01 AD-1955 1.06 1.04 AD-1955 1.12 1.05 AD-1955 1.11 1.14

TABLE 6 Serpina1 IC₅₀ Data IC50 24 hrs (nM) IC50 120 hrs (nM) Normalizedto Normalized Normalized Normalized Duplex ID AD-1955 to low dose toAD-1955 to low dose AD-44715.1 0.041 0.033 0.004 0.005 AD-44722.1 0.0970.075 0.006 0.008 AD-44734.1 0.237 0.237 0.008 0.01 AD-44717.1 0.1720.167 0.008 0.012 AD-44723.1 0.303 0.352 0.017 0.025 AD-44735.1 0.0820.076 0.011 0.01 AD-44724.1 0.086 0.098 0.003 0.005 AD-44719.1 0.3370.289 0.009 0.018 AD-44737.1 20.511 20.828 0.052 0.07

TABLE 7 Serpina1 IC₅₀ Data from Hep3B cells 24 hours after treatmentDuplex ID IC50 I (nM) IC50 II (nM) AD-44715.1 0.040 0.043 AD-44722.10.094 0.103 AD-44734.1 0.244 0.225 AD-44717.1 0.163 0.185 AD-44723.10.280 0.335 AD-44735.1 0.082 0.083 AD-44724.1 0.085 0.090 AD-44719.10.351 0.342 AD-44737.1 23.169 24.430Other embodiments are in the claims.

SEQ ID NO: 01: Human Serpina1 mRNA transcript variant 1 (NM_000295.4)   1 acaatgactc ctttcggtaa gtgcagtgga agctgtacac tgcccaggca aagcgtccgg  61 gcagcgtagg cgggcgactc agatcccagc cagtggactt agcccctgtt tgctcctccg 121 ataactgggg tgaccttggt taatattcac cagcagcctc ccccgttgcc cctctggatc 181 cactgcttaa atacggacga ggacagggcc ctgtctcctc agcttcaggc accaccactg 241 acctgggaca gtgaatcgac aatgccgtct tctgtctcgt ggggcatcct cctgctggca 301 ggcctgtgct gcctggtccc tgtctccctg gctgaggatc cccagggaga tgctgcccag 361 aagacagata catcccacca tgatcaggat cacccaacct tcaacaagat cacccccaac 421 ctggctgagt tcgccttcag cctataccgc cagctggcac accagtccaa cagcaccaat 481 atcttcttct ccccagtgag catcgctaca gcctttgcaa tgctctccct ggggaccaag 541 gctgacactc acgatgaaat cctggagggc ctgaatttca acctcacgga gattccggag 601 gctcagatcc atgaaggctt ccaggaactc ctccgtaccc tcaaccagcc agacagccag 661 ctccagctga ccaccggcaa tggcctgttc ctcagcgagg gcctgaagct agtggataag 721 tttttggagg atgttaaaaa gttgtaccac tcagaagcct tcactgtcaa cttcggggac 781 accgaagagg ccaagaaaca gatcaacgat tacgtggaga agggtactca agggaaaatt 841 gtggatttgg tcaaggagct tgacagagac acagtttttg ctctggtgaa ttacatcttc 901 tttaaaggca aatgggagag accctttgaa gtcaaggaca ccgaggaaga ggacttccac 961 gtggaccagg tgaccaccgt gaaggtgcct atgatgaagc gtttaggcat gtttaacatc1021 cagcactgta agaagctgtc cagctgggtg ctgctgatga aatacctggg caatgccacc1081 gccatcttct tcctgcctga tgaggggaaa ctacagcacc tggaaaatga actcacccac1141 gatatcatca ccaagttcct ggaaaatgaa gacagaaggt ctgccagctt acatttaccc1201 aaactgtcca ttactggaac ctatgatctg aagagcgtcc tgggtcaact gggcatcact1261 aaggtcttca gcaatggggc tgacctctcc ggggtcacag aggaggcacc cctgaagctc1321 tccaaggccg tgcataaggc tgtgctgacc atcgacgaga aagggactga agctgctggg1381 gccatgtttt tagaggccat acccatgtct atcccccccg aggtcaagtt caacaaaccc1441 tttgtcttct taatgattga acaaaatacc aagtctcccc tcttcatggg aaaagtggtg1501 aatcccaccc aaaaataact gcctctcgct cctcaacccc tcccctccat ccctggcccc1561 ctccctggat gacattaaag aagggttgag ctggtccctg cctgcatgtg actgtaaatc1621 cctcccatgt tttctctgag tctccctttg cctgctgagg ctgtatgtgg gctccaggta1681 acagtgctgt cttcgggccc cctgaactgt gttcatggag catctggctg ggtaggcaca1741 tgctgggctt gaatccaggg gggactgaat cctcagctta cggacctggg cccatctgtt1801 tctggagggc tccagtcttc cttgtcctgt cttggagtcc ccaagaagga atcacagggg1861 aggaaccaga taccagccat gaccccaggc tccaccaagc atcttcatgt ccccctgctc1921 atcccccact cccccccacc cagagttgct catcctgcca gggctggctg tgcccacccc1981 aaggctgccc tcctgggggc cccagaactg cctgatcgtg ccgtggccca gttttgtggc2041 atctgcagca acacaagaga gaggacaatg tcctcctctt gacccgctgt cacctaacca2101 gactcgggcc ctgcacctct caggcacttc tggaaaatga ctgaggcaga ttcttcctga2161 agcccattct ccatggggca acaaggacac ctattctgtc cttgtccttc catcgctgcc2221 ccagaaagcc tcacatatct ccgtttagaa tcaggtccct tctccccaga tgaagaggag2281 ggtctctgct ttgttttctc tatctcctcc tcagacttga ccaggcccag caggccccag2341 aagaccatta ccctatatcc cttctcctcc ctagtcacat ggccataggc ctgctgatgg2401 ctcaggaagg ccattgcaag gactcctcag ctatgggaga ggaagcacat cacccattga2461 cccccgcaac ccctcccttt cctcctctga gtcccgactg gggccacatg cagcctgact2521 tctttgtgcc tgttgctgtc cctgcagtct tcagagggcc accgcagctc cagtgccacg2581 gcaggaggct gttcctgaat agcccctgtg gtaagggcca ggagagtcct tccatcctcc2641 aaggccctgc taaaggacac agcagccagg aagtcccctg ggcccctagc tgaaggacag2701 cctgctccct ccgtctctac caggaatggc cttgtcctat ggaaggcact gccccatccc2761 aaactaatct aggaatcact gtctaaccac tcactgtcat gaatgtgtac ttaaaggatg2821 aggttgagtc ataccaaata gtgatttcga tagttcaaaa tggtgaaatt agcaattcta2881 catgattcag tctaatcaat ggataccgac tgtttcccac acaagtctcc tgttctctta2941 agcttactca ctgacagcct ttcactctcc acaaatacat taaagatatg gccatcacca3001 agccccctag gatgacacca gacctgagag tctgaagacc tggatccaag ttctgacttt3061 tccccctgac agctgtgtga ccttcgtgaa gtcgccaaac ctctctgagc cccagtcatt3121 gctagtaaga cctgcctttg agttggtatg atgttcaagt tagataacaa aatgtttata3181 cccattagaa cagagaataa atagaactac atttcttgcaSEQ ID NO: 02: Human Serpina1 mRNA, transcript variant 3 (NM_001002235.2)   1 tgggcaggaa ctgggcactg tgcccagggc atgcactgcc tccacgcagc aaccctcaga  61 gtcctgagct gaaccaagaa ggaggagggg gtcgggcctc cgaggaaggc ctagccgctg 121 ctgctgccag gaattccagg ttggaggggc ggcaacctcc tgccagcctt caggccactc 181 tcctgtgcct gccagaagag acagagcttg aggagagctt gaggagagca ggaaaggaca 241 atgccgtctt ctgtctcgtg gggcatcctc ctgctggcag gcctgtgctg cctggtccct 301 gtctccctgg ctgaggatcc ccagggagat gctgcccaga agacagatac atcccaccat 361 gatcaggatc acccaacctt caacaagatc acccccaacc tggctgagtt cgccttcagc 421 ctataccgcc agctggcaca ccagtccaac agcaccaata tcttcttctc cccagtgagc 481 atcgctacag cctttgcaat gctctccctg gggaccaagg ctgacactca cgatgaaatc 541 ctggagggcc tgaatttcaa cctcacggag attccggagg ctcagatcca tgaaggcttc 601 caggaactcc tccgtaccct caaccagcca gacagccagc tccagctgac caccggcaat 661 ggcctgttcc tcagcgaggg cctgaagcta gtggataagt ttttggagga tgttaaaaag 721 ttgtaccact cagaagcctt cactgtcaac ttcggggaca ccgaagaggc caagaaacag 781 atcaacgatt acgtggagaa gggtactcaa gggaaaattg tggatttggt caaggagctt 841 gacagagaca cagtttttgc tctggtgaat tacatcttct ttaaaggcaa atgggagaga 901 ccctttgaag tcaaggacac cgaggaagag gacttccacg tggaccaggt gaccaccgtg 961 aaggtgccta tgatgaagcg tttaggcatg tttaacatcc agcactgtaa gaagctgtcc1021 agctgggtgc tgctgatgaa atacctgggc aatgccaccg ccatcttctt cctgcctgat1081 gaggggaaac tacagcacct ggaaaatgaa ctcacccacg atatcatcac caagttcctg1141 gaaaatgaag acagaaggtc tgccagctta catttaccca aactgtccat tactggaacc1201 tatgatctga agagcgtcct gggtcaactg ggcatcacta aggtcttcag caatggggct1261 gacctctccg gggtcacaga ggaggcaccc ctgaagctct ccaaggccgt gcataaggct1321 gtgctgacca tcgacgagaa agggactgaa gctgctgggg ccatgttttt agaggccata1381 cccatgtcta tcccccccga ggtcaagttc aacaaaccct ttgtcttctt aatgattgaa1441 caaaatacca agtctcccct cttcatggga aaagtggtga atcccaccca aaaataactg1501 cctctcgctc ctcaacccct cccctccatc cctggccccc tccctggatg acattaaaga1561 agggttgagc tggtccctgc ctgcatgtga ctgtaaatcc ctcccatgtt ttctctgagt1621 ctccctttgc ctgctgaggc tgtatgtggg ctccaggtaa cagtgctgtc ttcgggcccc1681 ctgaactgtg ttcatggagc atctggctgg gtaggcacat gctgggcttg aatccagggg1741 ggactgaatc ctcagcttac ggacctgggc ccatctgttt ctggagggct ccagtcttcc1801 ttgtcctgtc ttggagtccc caagaaggaa tcacagggga ggaaccagat accagccatg1861 accccaggct ccaccaagca tcttcatgtc cccctgctca tcccccactc ccccccaccc1921 agagttgctc atcctgccag ggctggctgt gcccacccca aggctgccct cctgggggcc1981 ccagaactgc ctgatcgtgc cgtggcccag ttttgtggca tctgcagcaa cacaagagag2041 aggacaatgt cctcctcttg acccgctgtc acctaaccag actcgggccc tgcacctctc2101 aggcacttct ggaaaatgac tgaggcagat tcttcctgaa gcccattctc catggggcaa2161 caaggacacc tattctgtcc ttgtccttcc atcgctgccc cagaaagcct cacatatctc2221 cgtttagaat caggtccctt ctccccagat gaagaggagg gtctctgctt tgttttctct2281 atctcctcct cagacttgac caggcccagc aggccccaga agaccattac cctatatccc2341 ttctcctccc tagtcacatg gccataggcc tgctgatggc tcaggaaggc cattgcaagg2401 actcctcagc tatgggagag gaagcacatc acccattgac ccccgcaacc cctccctttc2461 ctcctctgag tcccgactgg ggccacatgc agcctgactt ctttgtgcct gttgctgtcc2521 ctgcagtctt cagagggcca ccgcagctcc agtgccacgg caggaggctg ttcctgaata2581 gcccctgtgg taagggccag gagagtcctt ccatcctcca aggccctgct aaaggacaca2641 gcagccagga agtcccctgg gcccctagct gaaggacagc ctgctccctc cgtctctacc2701 aggaatggcc ttgtcctatg gaaggcactg ccccatccca aactaatcta ggaatcactg2761 tctaaccact cactgtcatg aatgtgtact taaaggatga ggttgagtca taccaaatag2821 tgatttcgat agttcaaaat ggtgaaatta gcaattctac atgattcagt ctaatcaatg2881 gataccgact gtttcccaca caagtctcct gttctcttaa gcttactcac tgacagcctt2941 tcactctcca caaatacatt aaagatatgg ccatcaccaa gccccctagg atgacaccag3001 acctgagagt ctgaagacct ggatccaagt tctgactttt ccccctgaca gctgtgtgac3061 cttcgtgaag tcgccaaacc tctctgagcc ccagtcattg ctagtaagac ctgcctttga3121 gttggtatga tgttcaagtt agataacaaa atgtttatac ccattagaac agagaataaa3181 tagaactaca tttcttgcaSEQ ID NO: 03: Human Serpina1 mRNA transcript variant 2 (NM_001002236.2)   1 tgggcaggaa ctgggcactg tgcccagggc atgcactgcc tccacgcagc aaccctcaga  61 gtcctgagct gaaccaagaa ggaggagggg gtcgggcctc cgaggaaggc ctagccgctg 121 ctgctgccag gaattccagg ttggaggggc ggcaacctcc tgccagcctt caggccactc 181 tcctgtgcct gccagaagag acagagcttg aggagagctt gaggagagca ggaaagggcg 241 gcagtaagtc ttcagcatca ggcattttgg ggtgactcag taaatggtag atcttgctac 301 cagtggaaca gccactaagg attctgcagt gagagcagag ggccagctaa gtggtactct 361 cccagagact gtctgactca cgccaccccc tccaccttgg acacaggacg ctgtggtttc 421 tgagccaggt acaatgactc ctttcgcagc ctcccccgtt gcccctctgg atccactgct 481 taaatacgga cgaggacagg gccctgtctc ctcagcttca ggcaccacca ctgacctggg 541 acagtgaatc gacaatgccg tcttctgtct cgtggggcat cctcctgctg gcaggcctgt 601 gctgcctggt ccctgtctcc ctggctgagg atccccaggg agatgctgcc cagaagacag 661 atacatccca ccatgatcag gatcacccaa ccttcaacaa gatcaccccc aacctggctg 721 agttcgcctt cagcctatac cgccagctgg cacaccagtc caacagcacc aatatcttct 781 tctccccagt gagcatcgct acagcctttg caatgctctc cctggggacc aaggctgaca 841 ctcacgatga aatcctggag ggcctgaatt tcaacctcac ggagattccg gaggctcaga 901 tccatgaagg cttccaggaa ctcctccgta ccctcaacca gccagacagc cagctccagc 961 tgaccaccgg caatggcctg ttcctcagcg agggcctgaa gctagtggat aagtttttgg1021 aggatgttaa aaagttgtac cactcagaag ccttcactgt caacttcggg gacaccgaag1081 aggccaagaa acagatcaac gattacgtgg agaagggtac tcaagggaaa attgtggatt1141 tggtcaagga gcttgacaga gacacagttt ttgctctggt gaattacatc ttctttaaag1201 gcaaatggga gagacccttt gaagtcaagg acaccgagga agaggacttc cacgtggacc1261 aggtgaccac cgtgaaggtg cctatgatga agcgtttagg catgtttaac atccagcact1321 gtaagaagct gtccagctgg gtgctgctga tgaaatacct gggcaatgcc accgccatct1381 tcttcctgcc tgatgagggg aaactacagc acctggaaaa tgaactcacc cacgatatca1441 tcaccaagtt cctggaaaat gaagacagaa ggtctgccag cttacattta cccaaactgt1501 ccattactgg aacctatgat ctgaagagcg tcctgggtca actgggcatc actaaggtct1561 tcagcaatgg ggctgacctc tccggggtca cagaggaggc acccctgaag ctctccaagg1621 ccgtgcataa ggctgtgctg accatcgacg agaaagggac tgaagctgct ggggccatgt1681 ttttagaggc catacccatg tctatccccc ccgaggtcaa gttcaacaaa ccctttgtct1741 tcttaatgat tgaacaaaat accaagtctc ccctcttcat gggaaaagtg gtgaatccca1801 cccaaaaata actgcctctc gctcctcaac ccctcccctc catccctggc cccctccctg1861 gatgacatta aagaagggtt gagctggtcc ctgcctgcat gtgactgtaa atccctccca1921 tgttttctct gagtctccct ttgcctgctg aggctgtatg tgggctccag gtaacagtgc1981 tgtcttcggg ccccctgaac tgtgttcatg gagcatctgg ctgggtaggc acatgctggg2041 cttgaatcca ggggggactg aatcctcagc ttacggacct gggcccatct gtttctggag2101 ggctccagtc ttccttgtcc tgtcttggag tccccaagaa ggaatcacag gggaggaacc2161 agataccagc catgacccca ggctccacca agcatcttca tgtccccctg ctcatccccc2221 actccccccc acccagagtt gctcatcctg ccagggctgg ctgtgcccac cccaaggctg2281 ccctcctggg ggccccagaa ctgcctgatc gtgccgtggc ccagttttgt ggcatctgca2341 gcaacacaag agagaggaca atgtcctcct cttgacccgc tgtcacctaa ccagactcgg2401 gccctgcacc tctcaggcac ttctggaaaa tgactgaggc agattcttcc tgaagcccat2461 tctccatggg gcaacaagga cacctattct gtccttgtcc ttccatcgct gccccagaaa2521 gcctcacata tctccgttta gaatcaggtc ccttctcccc agatgaagag gagggtctct2581 gctttgtttt ctctatctcc tcctcagact tgaccaggcc cagcaggccc cagaagacca2641 ttaccctata tcccttctcc tccctagtca catggccata ggcctgctga tggctcagga2701 aggccattgc aaggactcct cagctatggg agaggaagca catcacccat tgacccccgc2761 aacccctccc tttcctcctc tgagtcccga ctggggccac atgcagcctg acttctttgt2821 gcctgttgct gtccctgcag tcttcagagg gccaccgcag ctccagtgcc acggcaggag2881 gctgttcctg aatagcccct gtggtaaggg ccaggagagt ccttccatcc tccaaggccc2941 tgctaaagga cacagcagcc aggaagtccc ctgggcccct agctgaagga cagcctgctc3001 cctccgtctc taccaggaat ggccttgtcc tatggaaggc actgccccat cccaaactaa3061 tctaggaatc actgtctaac cactcactgt catgaatgtg tacttaaagg atgaggttga3121 gtcataccaa atagtgattt cgatagttca aaatggtgaa attagcaatt ctacatgatt3181 cagtctaatc aatggatacc gactgtttcc cacacaagtc tcctgttctc ttaagcttac3241 tcactgacag cctttcactc tccacaaata cattaaagat atggccatca ccaagccccc3301 taggatgaca ccagacctga gagtctgaag acctggatcc aagttctgac ttttccccct3361 gacagctgtg tgaccttcgt gaagtcgcca aacctctctg agccccagtc attgctagta3421 agacctgcct ttgagttggt atgatgttca agttagataa caaaatgttt atacccatta3481 gaacagagaa taaatagaac tacatttctt gcaSEQ ID NO: 04: Human Serpina1 mRNA, transcript variant 4 (NM_001127700.1)   1 tgggcaggaa ctgggcactg tgcccagggc atgcactgcc tccacgcagc aaccctcaga  61 gtcctgagct gaaccaagaa ggaggagggg gtcgggcctc cgaggaaggc ctagccgctg 121 ctgctgccag gaattccagg ttggaggggc ggcaacctcc tgccagcctt caggccactc 181 tcctgtgcct gccagaagag acagagcttg aggagagctt gaggagagca ggaaaggtgg 241 gacattgctg ctgctgctca ctcagttcca caggacaatg ccgtcttctg tctcgtgggg 301 catcctcctg ctggcaggcc tgtgctgcct ggtccctgtc tccctggctg aggatcccca 361 gggagatgct gcccagaaga cagatacatc ccaccatgat caggatcacc caaccttcaa 421 caagatcacc cccaacctgg ctgagttcgc cttcagccta taccgccagc tggcacacca 481 gtccaacagc accaatatct tcttctcccc agtgagcatc gctacagcct ttgcaatgct 541 ctccctgggg accaaggctg acactcacga tgaaatcctg gagggcctga atttcaacct 601 cacggagatt ccggaggctc agatccatga aggcttccag gaactcctcc gtaccctcaa 661 ccagccagac agccagctcc agctgaccac cggcaatggc ctgttcctca gcgagggcct 721 gaagctagtg gataagtttt tggaggatgt taaaaagttg taccactcag aagccttcac 781 tgtcaacttc ggggacaccg aagaggccaa gaaacagatc aacgattacg tggagaaggg 841 tactcaaggg aaaattgtgg atttggtcaa ggagcttgac agagacacag tttttgctct 901 ggtgaattac atcttcttta aaggcaaatg ggagagaccc tttgaagtca aggacaccga 961 ggaagaggac ttccacgtgg accaggtgac caccgtgaag gtgcctatga tgaagcgttt1021 aggcatgttt aacatccagc actgtaagaa gctgtccagc tgggtgctgc tgatgaaata1081 cctgggcaat gccaccgcca tcttcttcct gcctgatgag gggaaactac agcacctgga1141 aaatgaactc acccacgata tcatcaccaa gttcctggaa aatgaagaca gaaggtctgc1201 cagcttacat ttacccaaac tgtccattac tggaacctat gatctgaaga gcgtcctggg1261 tcaactgggc atcactaagg tcttcagcaa tggggctgac ctctccgggg tcacagagga1321 ggcacccctg aagctctcca aggccgtgca taaggctgtg ctgaccatcg acgagaaagg1381 gactgaagct gctggggcca tgtttttaga ggccataccc atgtctatcc cccccgaggt1441 caagttcaac aaaccctttg tcttcttaat gattgaacaa aataccaagt ctcccctctt1501 catgggaaaa gtggtgaatc ccacccaaaa ataactgcct ctcgctcctc aacccctccc1561 ctccatccct ggccccctcc ctggatgaca ttaaagaagg gttgagctgg tccctgcctg1621 catgtgactg taaatccctc ccatgttttc tctgagtctc cctttgcctg ctgaggctgt1681 atgtgggctc caggtaacag tgctgtcttc gggccccctg aactgtgttc atggagcatc1741 tggctgggta ggcacatgct gggcttgaat ccagggggga ctgaatcctc agcttacgga1801 cctgggccca tctgtttctg gagggctcca gtcttccttg tcctgtcttg gagtccccaa1861 gaaggaatca caggggagga accagatacc agccatgacc ccaggctcca ccaagcatct1921 tcatgtcccc ctgctcatcc cccactcccc cccacccaga gttgctcatc ctgccagggc1981 tggctgtgcc caccccaagg ctgccctcct gggggcccca gaactgcctg atcgtgccgt2041 ggcccagttt tgtggcatct gcagcaacac aagagagagg acaatgtcct cctcttgacc2101 cgctgtcacc taaccagact cgggccctgc acctctcagg cacttctgga aaatgactga2161 ggcagattct tcctgaagcc cattctccat ggggcaacaa ggacacctat tctgtccttg2221 tccttccatc gctgccccag aaagcctcac atatctccgt ttagaatcag gtcccttctc2281 cccagatgaa gaggagggtc tctgctttgt tttctctatc tcctcctcag acttgaccag2341 gcccagcagg ccccagaaga ccattaccct atatcccttc tcctccctag tcacatggcc2401 ataggcctgc tgatggctca ggaaggccat tgcaaggact cctcagctat gggagaggaa2461 gcacatcacc cattgacccc cgcaacccct ccctttcctc ctctgagtcc cgactggggc2521 cacatgcagc ctgacttctt tgtgcctgtt gctgtccctg cagtcttcag agggccaccg2581 cagctccagt gccacggcag gaggctgttc ctgaatagcc cctgtggtaa gggccaggag2641 agtccttcca tcctccaagg ccctgctaaa ggacacagca gccaggaagt cccctgggcc2701 cctagctgaa ggacagcctg ctccctccgt ctctaccagg aatggccttg tcctatggaa2761 ggcactgccc catcccaaac taatctagga atcactgtct aaccactcac tgtcatgaat2821 gtgtacttaa aggatgaggt tgagtcatac caaatagtga tttcgatagt tcaaaatggt2881 gaaattagca attctacatg attcagtcta atcaatggat accgactgtt tcccacacaa2941 gtctcctgtt ctcttaagct tactcactga cagcctttca ctctccacaa atacattaaa3001 gatatggcca tcaccaagcc ccctaggatg acaccagacc tgagagtctg aagacctgga3061 tccaagttct gacttttccc cctgacagct gtgtgacctt cgtgaagtcg ccaaacctct3121 ctgagcccca gtcattgcta gtaagacctg cctttgagtt ggtatgatgt tcaagttaga3181 taacaaaatg tttataccca ttagaacaga gaataaatag aactacattt cttgcaSEQ ID NO: 05: Human Serpina1 mRNA, transcript variant 5 (NM_001127701.1)   1 tgggcaggaa ctgggcactg tgcccagggc atgcactgcc tccacgcagc aaccctcaga  61 gtcctgagct gaaccaagaa ggaggagggg gtcgggcctc cgaggaaggc ctagccgctg 121 ctgctgccag gaattccagg ttggaggggc ggcaacctcc tgccagcctt caggccactc 181 tcctgtgcct gccagaagag acagagcttg aggagagctt gaggagagca ggaaaggtgg 241 gacattgctg ctgctgctca ctcagttcca cagggcggca gtaagtcttc agcatcaggc 301 attttggggt gactcagtaa atggtagatc ttgctaccag tggaacagcc actaaggatt 361 ctgcagtgag agcagagggc cagctaagtg gtactctccc agagactgtc tgactcacgc 421 caccccctcc accttggaca caggacgctg tggtttctga gccagcagcc tcccccgttg 481 cccctctgga tccactgctt aaatacggac gaggacaggg ccctgtctcc tcagcttcag 541 gcaccaccac tgacctggga cagtgaatcg acaatgccgt cttctgtctc gtggggcatc 601 ctcctgctgg caggcctgtg ctgcctggtc cctgtctccc tggctgagga tccccaggga 661 gatgctgccc agaagacaga tacatcccac catgatcagg atcacccaac cttcaacaag 721 atcaccccca acctggctga gttcgccttc agcctatacc gccagctggc acaccagtcc 781 aacagcacca atatcttctt ctccccagtg agcatcgcta cagcctttgc aatgctctcc 841 ctggggacca aggctgacac tcacgatgaa atcctggagg gcctgaattt caacctcacg 901 gagattccgg aggctcagat ccatgaaggc ttccaggaac tcctccgtac cctcaaccag 961 ccagacagcc agctccagct gaccaccggc aatggcctgt tcctcagcga gggcctgaag1021 ctagtggata agtttttgga ggatgttaaa aagttgtacc actcagaagc cttcactgtc1081 aacttcgggg acaccgaaga ggccaagaaa cagatcaacg attacgtgga gaagggtact1141 caagggaaaa ttgtggattt ggtcaaggag cttgacagag acacagtttt tgctctggtg1201 aattacatct tctttaaagg caaatgggag agaccctttg aagtcaagga caccgaggaa1261 gaggacttcc acgtggacca ggtgaccacc gtgaaggtgc ctatgatgaa gcgtttaggc1321 atgtttaaca tccagcactg taagaagctg tccagctggg tgctgctgat gaaatacctg1381 ggcaatgcca ccgccatctt cttcctgcct gatgagggga aactacagca cctggaaaat1441 gaactcaccc acgatatcat caccaagttc ctggaaaatg aagacagaag gtctgccagc1501 ttacatttac ccaaactgtc cattactgga acctatgatc tgaagagcgt cctgggtcaa1561 ctgggcatca ctaaggtctt cagcaatggg gctgacctct ccggggtcac agaggaggca1621 cccctgaagc tctccaaggc cgtgcataag gctgtgctga ccatcgacga gaaagggact1681 gaagctgctg gggccatgtt tttagaggcc atacccatgt ctatcccccc cgaggtcaag1741 ttcaacaaac cctttgtctt cttaatgatt gaacaaaata ccaagtctcc cctcttcatg1801 ggaaaagtgg tgaatcccac ccaaaaataa ctgcctctcg ctcctcaacc cctcccctcc1861 atccctggcc ccctccctgg atgacattaa agaagggttg agctggtccc tgcctgcatg1921 tgactgtaaa tccctcccat gttttctctg agtctccctt tgcctgctga ggctgtatgt1981 gggctccagg taacagtgct gtcttcgggc cccctgaact gtgttcatgg agcatctggc2041 tgggtaggca catgctgggc ttgaatccag gggggactga atcctcagct tacggacctg2101 ggcccatctg tttctggagg gctccagtct tccttgtcct gtcttggagt ccccaagaag2161 gaatcacagg ggaggaacca gataccagcc atgaccccag gctccaccaa gcatcttcat2221 gtccccctgc tcatccccca ctccccccca cccagagttg ctcatcctgc cagggctggc2281 tgtgcccacc ccaaggctgc cctcctgggg gccccagaac tgcctgatcg tgccgtggcc2341 cagttttgtg gcatctgcag caacacaaga gagaggacaa tgtcctcctc ttgacccgct2401 gtcacctaac cagactcggg ccctgcacct ctcaggcact tctggaaaat gactgaggca2461 gattcttcct gaagcccatt ctccatgggg caacaaggac acctattctg tccttgtcct2521 tccatcgctg ccccagaaag cctcacatat ctccgtttag aatcaggtcc cttctcccca2581 gatgaagagg agggtctctg ctttgttttc tctatctcct cctcagactt gaccaggccc2641 agcaggcccc agaagaccat taccctatat cccttctcct ccctagtcac atggccatag2701 gcctgctgat ggctcaggaa ggccattgca aggactcctc agctatggga gaggaagcac2761 atcacccatt gacccccgca acccctccct ttcctcctct gagtcccgac tggggccaca2821 tgcagcctga cttctttgtg cctgttgctg tccctgcagt cttcagaggg ccaccgcagc2881 tccagtgcca cggcaggagg ctgttcctga atagcccctg tggtaagggc caggagagtc2941 cttccatcct ccaaggccct gctaaaggac acagcagcca ggaagtcccc tgggccccta3001 gctgaaggac agcctgctcc ctccgtctct accaggaatg gccttgtcct atggaaggca3061 ctgccccatc ccaaactaat ctaggaatca ctgtctaacc actcactgtc atgaatgtgt3121 acttaaagga tgaggttgag tcataccaaa tagtgatttc gatagttcaa aatggtgaaa3181 ttagcaattc tacatgattc agtctaatca atggataccg actgtttccc acacaagtct3241 cctgttctct taagcttact cactgacagc ctttcactct ccacaaatac attaaagata3301 tggccatcac caagccccct aggatgacac cagacctgag agtctgaaga cctggatcca3361 agttctgact tttccccctg acagctgtgt gaccttcgtg aagtcgccaa acctctctga3421 gccccagtca ttgctagtaa gacctgcctt tgagttggta tgatgttcaa gttagataac3481 aaaatgttta tacccattag aacagagaat aaatagaact acatttcttg caSEQ ID NO: 06: Human Serpina1 mRNA, transcript variant 6 (NM_001127702.1)   1 tgggcaggaa ctgggcactg tgcccagggc atgcactgcc tccacgcagc aaccctcaga  61 gtcctgagct gaaccaagaa ggaggagggg gtcgggcctc cgaggaaggc ctagccgctg 121 ctgctgccag gaattccagg ttggaggggc ggcaacctcc tgccagcctt caggccactc 181 tcctgtgcct gccagaagag acagagcttg aggagagctt gaggagagca ggaaaggtgg 241 gacattgctg ctgctgctca ctcagttcca cagcagcctc ccccgttgcc cctctggatc 301 cactgcttaa atacggacga ggacagggcc ctgtctcctc agcttcaggc accaccactg 361 acctgggaca gtgaatcgac aatgccgtct tctgtctcgt ggggcatcct cctgctggca 421 ggcctgtgct gcctggtccc tgtctccctg gctgaggatc cccagggaga tgctgcccag 481 aagacagata catcccacca tgatcaggat cacccaacct tcaacaagat cacccccaac 541 ctggctgagt tcgccttcag cctataccgc cagctggcac accagtccaa cagcaccaat 601 atcttcttct ccccagtgag catcgctaca gcctttgcaa tgctctccct ggggaccaag 661 gctgacactc acgatgaaat cctggagggc ctgaatttca acctcacgga gattccggag 721 gctcagatcc atgaaggctt ccaggaactc ctccgtaccc tcaaccagcc agacagccag 781 ctccagctga ccaccggcaa tggcctgttc ctcagcgagg gcctgaagct agtggataag 841 tttttggagg atgttaaaaa gttgtaccac tcagaagcct tcactgtcaa cttcggggac 901 accgaagagg ccaagaaaca gatcaacgat tacgtggaga agggtactca agggaaaatt 961 gtggatttgg tcaaggagct tgacagagac acagtttttg ctctggtgaa ttacatcttc1021 tttaaaggca aatgggagag accctttgaa gtcaaggaca ccgaggaaga ggacttccac1081 gtggaccagg tgaccaccgt gaaggtgcct atgatgaagc gtttaggcat gtttaacatc1141 cagcactgta agaagctgtc cagctgggtg ctgctgatga aatacctggg caatgccacc1201 gccatcttct tcctgcctga tgaggggaaa ctacagcacc tggaaaatga actcacccac1261 gatatcatca ccaagttcct ggaaaatgaa gacagaaggt ctgccagctt acatttaccc1321 aaactgtcca ttactggaac ctatgatctg aagagcgtcc tgggtcaact gggcatcact1381 aaggtcttca gcaatggggc tgacctctcc ggggtcacag aggaggcacc cctgaagctc1441 tccaaggccg tgcataaggc tgtgctgacc atcgacgaga aagggactga agctgctggg1501 gccatgtttt tagaggccat acccatgtct atcccccccg aggtcaagtt caacaaaccc1561 tttgtcttct taatgattga acaaaatacc aagtctcccc tcttcatggg aaaagtggtg1621 aatcccaccc aaaaataact gcctctcgct cctcaacccc tcccctccat ccctggcccc1681 ctccctggat gacattaaag aagggttgag ctggtccctg cctgcatgtg actgtaaatc1741 cctcccatgt tttctctgag tctccctttg cctgctgagg ctgtatgtgg gctccaggta1801 acagtgctgt cttcgggccc cctgaactgt gttcatggag catctggctg ggtaggcaca1861 tgctgggctt gaatccaggg gggactgaat cctcagctta cggacctggg cccatctgtt1921 tctggagggc tccagtcttc cttgtcctgt cttggagtcc ccaagaagga atcacagggg1981 aggaaccaga taccagccat gaccccaggc tccaccaagc atcttcatgt ccccctgctc2041 atcccccact cccccccacc cagagttgct catcctgcca gggctggctg tgcccacccc2101 aaggctgccc tcctgggggc cccagaactg cctgatcgtg ccgtggccca gttttgtggc2161 atctgcagca acacaagaga gaggacaatg tcctcctctt gacccgctgt cacctaacca2221 gactcgggcc ctgcacctct caggcacttc tggaaaatga ctgaggcaga ttcttcctga2281 agcccattct ccatggggca acaaggacac ctattctgtc cttgtccttc catcgctgcc2341 ccagaaagcc tcacatatct ccgtttagaa tcaggtccct tctccccaga tgaagaggag2401 ggtctctgct ttgttttctc tatctcctcc tcagacttga ccaggcccag caggccccag2461 aagaccatta ccctatatcc cttctcctcc ctagtcacat ggccataggc ctgctgatgg2521 ctcaggaagg ccattgcaag gactcctcag ctatgggaga ggaagcacat cacccattga2581 cccccgcaac ccctcccttt cctcctctga gtcccgactg gggccacatg cagcctgact2641 tctttgtgcc tgttgctgtc cctgcagtct tcagagggcc accgcagctc cagtgccacg2701 gcaggaggct gttcctgaat agcccctgtg gtaagggcca ggagagtcct tccatcctcc2761 aaggccctgc taaaggacac agcagccagg aagtcccctg ggcccctagc tgaaggacag2821 cctgctccct ccgtctctac caggaatggc cttgtcctat ggaaggcact gccccatccc2881 aaactaatct aggaatcact gtctaaccac tcactgtcat gaatgtgtac ttaaaggatg2941 aggttgagtc ataccaaata gtgatttcga tagttcaaaa tggtgaaatt agcaattcta3001 catgattcag tctaatcaat ggataccgac tgtttcccac acaagtctcc tgttctctta3061 agcttactca ctgacagcct ttcactctcc acaaatacat taaagatatg gccatcacca3121 agccccctag gatgacacca gacctgagag tctgaagacc tggatccaag ttctgacttt3181 tccccctgac agctgtgtga ccttcgtgaa gtcgccaaac ctctctgagc cccagtcatt3241 gctagtaaga cctgcctttg agttggtatg atgttcaagt tagataacaa aatgtttata3301 cccattagaa cagagaataa atagaactac atttcttgcaSEQ ID NO: 07: Human Serpina1 mRNA, transcript variant 7 (NM_001127703.1)   1 tgggcaggaa ctgggcactg tgcccagggc atgcactgcc tccacgcagc aaccctcaga  61 gtcctgagct gaaccaagaa ggaggagggg gtcgggcctc cgaggaaggc ctagccgctg 121 ctgctgccag gaattccagg ttggaggggc ggcaacctcc tgccagcctt caggccactc 181 tcctgtgcct gccagaagag acagagcttg aggagagctt gaggagagca ggaaagggcg 241 gcagtaagtc ttcagcatca ggcattttgg ggtgactcag taaatggtag atcttgctac 301 cagtggaaca gccactaagg attctgcagt gagagcagag ggccagctaa gtggtactct 361 cccagagact gtctgactca cgccaccccc tccaccttgg acacaggacg ctgtggtttc 421 tgagccagca gcctcccccg ttgcccctct ggatccactg cttaaatacg gacgaggaca 481 gggccctgtc tcctcagctt caggcaccac cactgacctg ggacagtgaa tcgacaatgc 541 cgtcttctgt ctcgtggggc atcctcctgc tggcaggcct gtgctgcctg gtccctgtct 601 ccctggctga ggatccccag ggagatgctg cccagaagac agatacatcc caccatgatc 661 aggatcaccc aaccttcaac aagatcaccc ccaacctggc tgagttcgcc ttcagcctat 721 accgccagct ggcacaccag tccaacagca ccaatatctt cttctcccca gtgagcatcg 781 ctacagcctt tgcaatgctc tccctgggga ccaaggctga cactcacgat gaaatcctgg 841 agggcctgaa tttcaacctc acggagattc cggaggctca gatccatgaa ggcttccagg 901 aactcctccg taccctcaac cagccagaca gccagctcca gctgaccacc ggcaatggcc 961 tgttcctcag cgagggcctg aagctagtgg ataagttttt ggaggatgtt aaaaagttgt1021 accactcaga agccttcact gtcaacttcg gggacaccga agaggccaag aaacagatca1081 acgattacgt ggagaagggt actcaaggga aaattgtgga tttggtcaag gagcttgaca1141 gagacacagt ttttgctctg gtgaattaca tcttctttaa aggcaaatgg gagagaccct1201 ttgaagtcaa ggacaccgag gaagaggact tccacgtgga ccaggtgacc accgtgaagg1261 tgcctatgat gaagcgttta ggcatgttta acatccagca ctgtaagaag ctgtccagct1321 gggtgctgct gatgaaatac ctgggcaatg ccaccgccat cttcttcctg cctgatgagg1381 ggaaactaca gcacctggaa aatgaactca cccacgatat catcaccaag ttcctggaaa1441 atgaagacag aaggtctgcc agcttacatt tacccaaact gtccattact ggaacctatg1501 atctgaagag cgtcctgggt caactgggca tcactaaggt cttcagcaat ggggctgacc1561 tctccggggt cacagaggag gcacccctga agctctccaa ggccgtgcat aaggctgtgc1621 tgaccatcga cgagaaaggg actgaagctg ctggggccat gtttttagag gccataccca1681 tgtctatccc ccccgaggtc aagttcaaca aaccctttgt cttcttaatg attgaacaaa1741 ataccaagtc tcccctcttc atgggaaaag tggtgaatcc cacccaaaaa taactgcctc1801 tcgctcctca acccctcccc tccatccctg gccccctccc tggatgacat taaagaaggg1861 ttgagctggt ccctgcctgc atgtgactgt aaatccctcc catgttttct ctgagtctcc1921 ctttgcctgc tgaggctgta tgtgggctcc aggtaacagt gctgtcttcg ggccccctga1981 actgtgttca tggagcatct ggctgggtag gcacatgctg ggcttgaatc caggggggac2041 tgaatcctca gcttacggac ctgggcccat ctgtttctgg agggctccag tcttccttgt2101 cctgtcttgg agtccccaag aaggaatcac aggggaggaa ccagatacca gccatgaccc2161 caggctccac caagcatctt catgtccccc tgctcatccc ccactccccc ccacccagag2221 ttgctcatcc tgccagggct ggctgtgccc accccaaggc tgccctcctg ggggccccag2281 aactgcctga tcgtgccgtg gcccagtttt gtggcatctg cagcaacaca agagagagga2341 caatgtcctc ctcttgaccc gctgtcacct aaccagactc gggccctgca cctctcaggc2401 acttctggaa aatgactgag gcagattctt cctgaagccc attctccatg gggcaacaag2461 gacacctatt ctgtccttgt ccttccatcg ctgccccaga aagcctcaca tatctccgtt2521 tagaatcagg tcccttctcc ccagatgaag aggagggtct ctgctttgtt ttctctatct2581 cctcctcaga cttgaccagg cccagcaggc cccagaagac cattacccta tatcccttct2641 cctccctagt cacatggcca taggcctgct gatggctcag gaaggccatt gcaaggactc2701 ctcagctatg ggagaggaag cacatcaccc attgaccccc gcaacccctc cctttcctcc2761 tctgagtccc gactggggcc acatgcagcc tgacttcttt gtgcctgttg ctgtccctgc2821 agtcttcaga gggccaccgc agctccagtg ccacggcagg aggctgttcc tgaatagccc2881 ctgtggtaag ggccaggaga gtccttccat cctccaaggc cctgctaaag gacacagcag2941 ccaggaagtc ccctgggccc ctagctgaag gacagcctgc tccctccgtc tctaccagga3001 atggccttgt cctatggaag gcactgcccc atcccaaact aatctaggaa tcactgtcta3061 accactcact gtcatgaatg tgtacttaaa ggatgaggtt gagtcatacc aaatagtgat3121 ttcgatagtt caaaatggtg aaattagcaa ttctacatga ttcagtctaa tcaatggata3181 ccgactgttt cccacacaag tctcctgttc tcttaagctt actcactgac agcctttcac3241 tctccacaaa tacattaaag atatggccat caccaagccc cctaggatga caccagacct3301 gagagtctga agacctggat ccaagttctg acttttcccc ctgacagctg tgtgaccttc3361 gtgaagtcgc caaacctctc tgagccccag tcattgctag taagacctgc ctttgagttg3421 gtatgatgtt caagttagat aacaaaatgt ttatacccat tagaacagag aataaataga3481 actacatttc ttgcaSEQ ID NO: 08: Human Serpina1 mRNA, transcript variant 8 (NM_001127704.1)   1 tgggcaggaa ctgggcactg tgcccagggc atgcactgcc tccacgcagc aaccctcaga  61 gtcctgagct gaaccaagaa ggaggagggg gtcgggcctc cgaggaaggc ctagccgctg 121 ctgctgccag gaattccagg ttggaggggc ggcaacctcc tgccagcctt caggccactc 181 tcctgtgcct gccagaagag acagagcttg aggagagctt gaggagagca ggaaagggcg 241 gcagtaagtc ttcagcatca ggcattttgg ggtgactcag taaatggtag atcttgctac 301 cagtggaaca gccactaagg attctgcagt gagagcagag ggccagctaa gtggtactct 361 cccagagact gtctgactca cgccaccccc tccaccttgg acacaggacg ctgtggtttc 421 tgagccagcc tcccccgttg cccctctgga tccactgctt aaatacggac gaggacaggg 481 ccctgtctcc tcagcttcag gcaccaccac tgacctggga cagtgaatcg acaatgccgt 541 cttctgtctc gtggggcatc ctcctgctgg caggcctgtg ctgcctggtc cctgtctccc 601 tggctgagga tccccaggga gatgctgccc agaagacaga tacatcccac catgatcagg 661 atcacccaac cttcaacaag atcaccccca acctggctga gttcgccttc agcctatacc 721 gccagctggc acaccagtcc aacagcacca atatcttctt ctccccagtg agcatcgcta 781 cagcctttgc aatgctctcc ctggggacca aggctgacac tcacgatgaa atcctggagg 841 gcctgaattt caacctcacg gagattccgg aggctcagat ccatgaaggc ttccaggaac 901 tcctccgtac cctcaaccag ccagacagcc agctccagct gaccaccggc aatggcctgt 961 tcctcagcga gggcctgaag ctagtggata agtttttgga ggatgttaaa aagttgtacc1021 actcagaagc cttcactgtc aacttcgggg acaccgaaga ggccaagaaa cagatcaacg1081 attacgtgga gaagggtact caagggaaaa ttgtggattt ggtcaaggag cttgacagag1141 acacagtttt tgctctggtg aattacatct tctttaaagg caaatgggag agaccctttg1201 aagtcaagga caccgaggaa gaggacttcc acgtggacca ggtgaccacc gtgaaggtgc1261 ctatgatgaa gcgtttaggc atgtttaaca tccagcactg taagaagctg tccagctggg1321 tgctgctgat gaaatacctg ggcaatgcca ccgccatctt cttcctgcct gatgagggga1381 aactacagca cctggaaaat gaactcaccc acgatatcat caccaagttc ctggaaaatg1441 aagacagaag gtctgccagc ttacatttac ccaaactgtc cattactgga acctatgatc1501 tgaagagcgt cctgggtcaa ctgggcatca ctaaggtctt cagcaatggg gctgacctct1561 ccggggtcac agaggaggca cccctgaagc tctccaaggc cgtgcataag gctgtgctga1621 ccatcgacga gaaagggact gaagctgctg gggccatgtt tttagaggcc atacccatgt1681 ctatcccccc cgaggtcaag ttcaacaaac cctttgtctt cttaatgatt gaacaaaata1741 ccaagtctcc cctcttcatg ggaaaagtgg tgaatcccac ccaaaaataa ctgcctctcg1801 ctcctcaacc cctcccctcc atccctggcc ccctccctgg atgacattaa agaagggttg1861 agctggtccc tgcctgcatg tgactgtaaa tccctcccat gttttctctg agtctccctt1921 tgcctgctga ggctgtatgt gggctccagg taacagtgct gtcttcgggc cccctgaact1981 gtgttcatgg agcatctggc tgggtaggca catgctgggc ttgaatccag gggggactga2041 atcctcagct tacggacctg ggcccatctg tttctggagg gctccagtct tccttgtcct2101 gtcttggagt ccccaagaag gaatcacagg ggaggaacca gataccagcc atgaccccag2161 gctccaccaa gcatcttcat gtccccctgc tcatccccca ctccccccca cccagagttg2221 ctcatcctgc cagggctggc tgtgcccacc ccaaggctgc cctcctgggg gccccagaac2281 tgcctgatcg tgccgtggcc cagttttgtg gcatctgcag caacacaaga gagaggacaa2341 tgtcctcctc ttgacccgct gtcacctaac cagactcggg ccctgcacct ctcaggcact2401 tctggaaaat gactgaggca gattcttcct gaagcccatt ctccatgggg caacaaggac2461 acctattctg tccttgtcct tccatcgctg ccccagaaag cctcacatat ctccgtttag2521 aatcaggtcc cttctcccca gatgaagagg agggtctctg ctttgttttc tctatctcct2581 cctcagactt gaccaggccc agcaggcccc agaagaccat taccctatat cccttctcct2641 ccctagtcac atggccatag gcctgctgat ggctcaggaa ggccattgca aggactcctc2701 agctatggga gaggaagcac atcacccatt gacccccgca acccctccct ttcctcctct2761 gagtcccgac tggggccaca tgcagcctga cttctttgtg cctgttgctg tccctgcagt2821 cttcagaggg ccaccgcagc tccagtgcca cggcaggagg ctgttcctga atagcccctg2881 tggtaagggc caggagagtc cttccatcct ccaaggccct gctaaaggac acagcagcca2941 ggaagtcccc tgggccccta gctgaaggac agcctgctcc ctccgtctct accaggaatg3001 gccttgtcct atggaaggca ctgccccatc ccaaactaat ctaggaatca ctgtctaacc3061 actcactgtc atgaatgtgt acttaaagga tgaggttgag tcataccaaa tagtgatttc3121 gatagttcaa aatggtgaaa ttagcaattc tacatgattc agtctaatca atggataccg3181 actgtttccc acacaagtct cctgttctct taagcttact cactgacagc ctttcactct3241 ccacaaatac attaaagata tggccatcac caagccccct aggatgacac cagacctgag3301 agtctgaaga cctggatcca agttctgact tttccccctg acagctgtgt gaccttcgtg3361 aagtcgccaa acctctctga gccccagtca ttgctagtaa gacctgcctt tgagttggta3421 tgatgttcaa gttagataac aaaatgttta tacccattag aacagagaat aaatagaact3481 acatttcttg caSEQ ID NO: 09: Human Serpina1 mRNA, transcript variant 9 (NM_001127705.1)   1 tgggcaggaa ctgggcactg tgcccagggc atgcactgcc tccacgcagc aaccctcaga  61 gtcctgagct gaaccaagaa ggaggagggg gtcgggcctc cgaggaaggc ctagccgctg 121 ctgctgccag gaattccagg ttggaggggc ggcaacctcc tgccagcctt caggccactc 181 tcctgtgcct gccagaagag acagagcttg aggagagctt gaggagagca ggaaagggcg 241 gcagtaagtc ttcagcatca ggcattttgg ggtgactcag taaatggtag atcttgctac 301 cagtggaaca gccactaagg attctgcagt gagagcagag ggccagctaa gtggtactct 361 cccagagact gtctgactca cgccaccccc tccaccttgg acacaggacg ctgtggtttc 421 tgagccaggt acaatgactc ctttcgcctc ccccgttgcc cctctggatc cactgcttaa 481 atacggacga ggacagggcc ctgtctcctc agcttcaggc accaccactg acctgggaca 541 gtgaatcgac aatgccgtct tctgtctcgt ggggcatcct cctgctggca ggcctgtgct 601 gcctggtccc tgtctccctg gctgaggatc cccagggaga tgctgcccag aagacagata 661 catcccacca tgatcaggat cacccaacct tcaacaagat cacccccaac ctggctgagt 721 tcgccttcag cctataccgc cagctggcac accagtccaa cagcaccaat atcttcttct 781 ccccagtgag catcgctaca gcctttgcaa tgctctccct ggggaccaag gctgacactc 841 acgatgaaat cctggagggc ctgaatttca acctcacgga gattccggag gctcagatcc 901 atgaaggctt ccaggaactc ctccgtaccc tcaaccagcc agacagccag ctccagctga 961 ccaccggcaa tggcctgttc ctcagcgagg gcctgaagct agtggataag tttttggagg1021 atgttaaaaa gttgtaccac tcagaagcct tcactgtcaa cttcggggac accgaagagg1081 ccaagaaaca gatcaacgat tacgtggaga agggtactca agggaaaatt gtggatttgg1141 tcaaggagct tgacagagac acagtttttg ctctggtgaa ttacatcttc tttaaaggca1201 aatgggagag accctttgaa gtcaaggaca ccgaggaaga ggacttccac gtggaccagg1261 tgaccaccgt gaaggtgcct atgatgaagc gtttaggcat gtttaacatc cagcactgta1321 agaagctgtc cagctgggtg ctgctgatga aatacctggg caatgccacc gccatcttct1381 tcctgcctga tgaggggaaa ctacagcacc tggaaaatga actcacccac gatatcatca1441 ccaagttcct ggaaaatgaa gacagaaggt ctgccagctt acatttaccc aaactgtcca1501 ttactggaac ctatgatctg aagagcgtcc tgggtcaact gggcatcact aaggtcttca1561 gcaatggggc tgacctctcc ggggtcacag aggaggcacc cctgaagctc tccaaggccg1621 tgcataaggc tgtgctgacc atcgacgaga aagggactga agctgctggg gccatgtttt1681 tagaggccat acccatgtct atcccccccg aggtcaagtt caacaaaccc tttgtcttct1741 taatgattga acaaaatacc aagtctcccc tcttcatggg aaaagtggtg aatcccaccc1801 aaaaataact gcctctcgct cctcaacccc tcccctccat ccctggcccc ctccctggat1861 gacattaaag aagggttgag ctggtccctg cctgcatgtg actgtaaatc cctcccatgt1921 tttctctgag tctccctttg cctgctgagg ctgtatgtgg gctccaggta acagtgctgt1981 cttcgggccc cctgaactgt gttcatggag catctggctg ggtaggcaca tgctgggctt2041 gaatccaggg gggactgaat cctcagctta cggacctggg cccatctgtt tctggagggc2101 tccagtcttc cttgtcctgt cttggagtcc ccaagaagga atcacagggg aggaaccaga2161 taccagccat gaccccaggc tccaccaagc atcttcatgt ccccctgctc atcccccact2221 cccccccacc cagagttgct catcctgcca gggctggctg tgcccacccc aaggctgccc2281 tcctgggggc cccagaactg cctgatcgtg ccgtggccca gttttgtggc atctgcagca2341 acacaagaga gaggacaatg tcctcctctt gacccgctgt cacctaacca gactcgggcc2401 ctgcacctct caggcacttc tggaaaatga ctgaggcaga ttcttcctga agcccattct2461 ccatggggca acaaggacac ctattctgtc cttgtccttc catcgctgcc ccagaaagcc2521 tcacatatct ccgtttagaa tcaggtccct tctccccaga tgaagaggag ggtctctgct2581 ttgttttctc tatctcctcc tcagacttga ccaggcccag caggccccag aagaccatta2641 ccctatatcc cttctcctcc ctagtcacat ggccataggc ctgctgatgg ctcaggaagg2701 ccattgcaag gactcctcag ctatgggaga ggaagcacat cacccattga cccccgcaac2761 ccctcccttt cctcctctga gtcccgactg gggccacatg cagcctgact tctttgtgcc2821 tgttgctgtc cctgcagtct tcagagggcc accgcagctc cagtgccacg gcaggaggct2881 gttcctgaat agcccctgtg gtaagggcca ggagagtcct tccatcctcc aaggccctgc2941 taaaggacac agcagccagg aagtcccctg ggcccctagc tgaaggacag cctgctccct3001 ccgtctctac caggaatggc cttgtcctat ggaaggcact gccccatccc aaactaatct3061 aggaatcact gtctaaccac tcactgtcat gaatgtgtac ttaaaggatg aggttgagtc3121 ataccaaata gtgatttcga tagttcaaaa tggtgaaatt agcaattcta catgattcag3181 tctaatcaat ggataccgac tgtttcccac acaagtctcc tgttctctta agcttactca3241 ctgacagcct ttcactctcc acaaatacat taaagatatg gccatcacca agccccctag3301 gatgacacca gacctgagag tctgaagacc tggatccaag ttctgacttt tccccctgac3361 agctgtgtga ccttcgtgaa gtcgccaaac ctctctgagc cccagtcatt gctagtaaga3421 cctgcctttg agttggtatg atgttcaagt tagataacaa aatgtttata cccattagaa3481 cagagaataa atagaactac atttcttgcaSEQ ID NO: 10: Human Serpina1 mRNA, transcript variant 10 (NM_001127706.1)   1 tgggcaggaa ctgggcactg tgcccagggc atgcactgcc tccacgcagc aaccctcaga  61 gtcctgagct gaaccaagaa ggaggagggg gtcgggcctc cgaggaaggc ctagccgctg 121 ctgctgccag gaattccagg ttggaggggc ggcaacctcc tgccagcctt caggccactc 181 tcctgtgcct gccagaagag acagagcttg aggagagctt gaggagagca ggaaagcagc 241 ctcccccgtt gcccctctgg atccactgct taaatacgga cgaggacagg gccctgtctc 301 ctcagcttca ggcaccacca ctgacctggg acagtgaatc gacaatgccg tcttctgtct 361 cgtggggcat cctcctgctg gcaggcctgt gctgcctggt ccctgtctcc ctggctgagg 421 atccccaggg agatgctgcc cagaagacag atacatccca ccatgatcag gatcacccaa 481 ccttcaacaa gatcaccccc aacctggctg agttcgcctt cagcctatac cgccagctgg 541 cacaccagtc caacagcacc aatatcttct tctccccagt gagcatcgct acagcctttg 601 caatgctctc cctggggacc aaggctgaca ctcacgatga aatcctggag ggcctgaatt 661 tcaacctcac ggagattccg gaggctcaga tccatgaagg cttccaggaa ctcctccgta 721 ccctcaacca gccagacagc cagctccagc tgaccaccgg caatggcctg ttcctcagcg 781 agggcctgaa gctagtggat aagtttttgg aggatgttaa aaagttgtac cactcagaag 841 ccttcactgt caacttcggg gacaccgaag aggccaagaa acagatcaac gattacgtgg 901 agaagggtac tcaagggaaa attgtggatt tggtcaagga gcttgacaga gacacagttt 961 ttgctctggt gaattacatc ttctttaaag gcaaatggga gagacccttt gaagtcaagg1021 acaccgagga agaggacttc cacgtggacc aggtgaccac cgtgaaggtg cctatgatga1081 agcgtttagg catgtttaac atccagcact gtaagaagct gtccagctgg gtgctgctga1141 tgaaatacct gggcaatgcc accgccatct tcttcctgcc tgatgagggg aaactacagc1201 acctggaaaa tgaactcacc cacgatatca tcaccaagtt cctggaaaat gaagacagaa1261 ggtctgccag cttacattta cccaaactgt ccattactgg aacctatgat ctgaagagcg1321 tcctgggtca actgggcatc actaaggtct tcagcaatgg ggctgacctc tccggggtca1381 cagaggaggc acccctgaag ctctccaagg ccgtgcataa ggctgtgctg accatcgacg1441 agaaagggac tgaagctgct ggggccatgt ttttagaggc catacccatg tctatccccc1501 ccgaggtcaa gttcaacaaa ccctttgtct tcttaatgat tgaacaaaat accaagtctc1561 ccctcttcat gggaaaagtg gtgaatccca cccaaaaata actgcctctc gctcctcaac1621 ccctcccctc catccctggc cccctccctg gatgacatta aagaagggtt gagctggtcc1681 ctgcctgcat gtgactgtaa atccctccca tgttttctct gagtctccct ttgcctgctg1741 aggctgtatg tgggctccag gtaacagtgc tgtcttcggg ccccctgaac tgtgttcatg1801 gagcatctgg ctgggtaggc acatgctggg cttgaatcca ggggggactg aatcctcagc1861 ttacggacct gggcccatct gtttctggag ggctccagtc ttccttgtcc tgtcttggag1921 tccccaagaa ggaatcacag gggaggaacc agataccagc catgacccca ggctccacca1981 agcatcttca tgtccccctg ctcatccccc actccccccc acccagagtt gctcatcctg2041 ccagggctgg ctgtgcccac cccaaggctg ccctcctggg ggccccagaa ctgcctgatc2101 gtgccgtggc ccagttttgt ggcatctgca gcaacacaag agagaggaca atgtcctcct2161 cttgacccgc tgtcacctaa ccagactcgg gccctgcacc tctcaggcac ttctggaaaa2221 tgactgaggc agattcttcc tgaagcccat tctccatggg gcaacaagga cacctattct2281 gtccttgtcc ttccatcgct gccccagaaa gcctcacata tctccgttta gaatcaggtc2341 ccttctcccc agatgaagag gagggtctct gctttgtttt ctctatctcc tcctcagact2401 tgaccaggcc cagcaggccc cagaagacca ttaccctata tcccttctcc tccctagtca2461 catggccata ggcctgctga tggctcagga aggccattgc aaggactcct cagctatggg2521 agaggaagca catcacccat tgacccccgc aacccctccc tttcctcctc tgagtcccga2581 ctggggccac atgcagcctg acttctttgt gcctgttgct gtccctgcag tcttcagagg2641 gccaccgcag ctccagtgcc acggcaggag gctgttcctg aatagcccct gtggtaaggg2701 ccaggagagt ccttccatcc tccaaggccc tgctaaagga cacagcagcc aggaagtccc2761 ctgggcccct agctgaagga cagcctgctc cctccgtctc taccaggaat ggccttgtcc2821 tatggaaggc actgccccat cccaaactaa tctaggaatc actgtctaac cactcactgt2881 catgaatgtg tacttaaagg atgaggttga gtcataccaa atagtgattt cgatagttca2941 aaatggtgaa attagcaatt ctacatgatt cagtctaatc aatggatacc gactgtttcc3001 cacacaagtc tcctgttctc ttaagcttac tcactgacag cctttcactc tccacaaata3061 cattaaagat atggccatca ccaagccccc taggatgaca ccagacctga gagtctgaag3121 acctggatcc aagttctgac ttttccccct gacagctgtg tgaccttcgt gaagtcgcca3181 aacctctctg agccccagtc attgctagta agacctgcct ttgagttggt atgatgttca3241 agttagataa caaaatgttt atacccatta gaacagagaa taaatagaac tacatttctt3301 gcaSEQ ID NO: 11: Human Serpina1 mRNA, transcript variant 11 (NM_001127707.1)   1 tgggcaggaa ctgggcactg tgcccagggc atgcactgcc tccacgcagc aaccctcaga  61 gtcctgagct gaaccaagaa ggaggagggg gtcgggcctc cgaggaaggc ctagccgctg 121 ctgctgccag gaattccagg ttggaggggc ggcaacctcc tgccagcctt caggccactc 181 tcctgtgcct gccagaagag acagagcttg aggagagctt gaggagagca ggaaagcctc 241 ccccgttgcc cctctggatc cactgcttaa atacggacga ggacagggcc ctgtctcctc 301 agcttcaggc accaccactg acctgggaca gtgaatcgac aatgccgtct tctgtctcgt 361 ggggcatcct cctgctggca ggcctgtgct gcctggtccc tgtctccctg gctgaggatc 421 cccagggaga tgctgcccag aagacagata catcccacca tgatcaggat cacccaacct 481 tcaacaagat cacccccaac ctggctgagt tcgccttcag cctataccgc cagctggcac 541 accagtccaa cagcaccaat atcttcttct ccccagtgag catcgctaca gcctttgcaa 601 tgctctccct ggggaccaag gctgacactc acgatgaaat cctggagggc ctgaatttca 661 acctcacgga gattccggag gctcagatcc atgaaggctt ccaggaactc ctccgtaccc 721 tcaaccagcc agacagccag ctccagctga ccaccggcaa tggcctgttc ctcagcgagg 781 gcctgaagct agtggataag tttttggagg atgttaaaaa gttgtaccac tcagaagcct 841 tcactgtcaa cttcggggac accgaagagg ccaagaaaca gatcaacgat tacgtggaga 901 agggtactca agggaaaatt gtggatttgg tcaaggagct tgacagagac acagtttttg 961 ctctggtgaa ttacatcttc tttaaaggca aatgggagag accctttgaa gtcaaggaca1021 ccgaggaaga ggacttccac gtggaccagg tgaccaccgt gaaggtgcct atgatgaagc1081 gtttaggcat gtttaacatc cagcactgta agaagctgtc cagctgggtg ctgctgatga1141 aatacctggg caatgccacc gccatcttct tcctgcctga tgaggggaaa ctacagcacc1201 tggaaaatga actcacccac gatatcatca ccaagttcct ggaaaatgaa gacagaaggt1261 ctgccagctt acatttaccc aaactgtcca ttactggaac ctatgatctg aagagcgtcc1321 tgggtcaact gggcatcact aaggtcttca gcaatggggc tgacctctcc ggggtcacag1381 aggaggcacc cctgaagctc tccaaggccg tgcataaggc tgtgctgacc atcgacgaga1441 aagggactga agctgctggg gccatgtttt tagaggccat acccatgtct atcccccccg1501 aggtcaagtt caacaaaccc tttgtcttct taatgattga acaaaatacc aagtctcccc1561 tcttcatggg aaaagtggtg aatcccaccc aaaaataact gcctctcgct cctcaacccc1621 tcccctccat ccctggcccc ctccctggat gacattaaag aagggttgag ctggtccctg1681 cctgcatgtg actgtaaatc cctcccatgt tttctctgag tctccctttg cctgctgagg1741 ctgtatgtgg gctccaggta acagtgctgt cttcgggccc cctgaactgt gttcatggag1801 catctggctg ggtaggcaca tgctgggctt gaatccaggg gggactgaat cctcagctta1861 cggacctggg cccatctgtt tctggagggc tccagtcttc cttgtcctgt cttggagtcc1921 ccaagaagga atcacagggg aggaaccaga taccagccat gaccccaggc tccaccaagc1981 atcttcatgt ccccctgctc atcccccact cccccccacc cagagttgct catcctgcca2041 gggctggctg tgcccacccc aaggctgccc tcctgggggc cccagaactg cctgatcgtg2101 ccgtggccca gttttgtggc atctgcagca acacaagaga gaggacaatg tcctcctctt2161 gacccgctgt cacctaacca gactcgggcc ctgcacctct caggcacttc tggaaaatga2221 ctgaggcaga ttcttcctga agcccattct ccatggggca acaaggacac ctattctgtc2281 cttgtccttc catcgctgcc ccagaaagcc tcacatatct ccgtttagaa tcaggtccct2341 tctccccaga tgaagaggag ggtctctgct ttgttttctc tatctcctcc tcagacttga2401 ccaggcccag caggccccag aagaccatta ccctatatcc cttctcctcc ctagtcacat2461 ggccataggc ctgctgatgg ctcaggaagg ccattgcaag gactcctcag ctatgggaga2521 ggaagcacat cacccattga cccccgcaac ccctcccttt cctcctctga gtcccgactg2581 gggccacatg cagcctgact tctttgtgcc tgttgctgtc cctgcagtct tcagagggcc2641 accgcagctc cagtgccacg gcaggaggct gttcctgaat agcccctgtg gtaagggcca2701 ggagagtcct tccatcctcc aaggccctgc taaaggacac agcagccagg aagtcccctg2761 ggcccctagc tgaaggacag cctgctccct ccgtctctac caggaatggc cttgtcctat2821 ggaaggcact gccccatccc aaactaatct aggaatcact gtctaaccac tcactgtcat2881 gaatgtgtac ttaaaggatg aggttgagtc ataccaaata gtgatttcga tagttcaaaa2941 tggtgaaatt agcaattcta catgattcag tctaatcaat ggataccgac tgtttcccac3001 acaagtctcc tgttctctta agcttactca ctgacagcct ttcactctcc acaaatacat3061 taaagatatg gccatcacca agccccctag gatgacacca gacctgagag tctgaagacc3121 tggatccaag ttctgacttt tccccctgac agctgtgtga ccttcgtgaa gtcgccaaac3181 ctctctgagc cccagtcatt gctagtaaga cctgcctttg agttggtatg atgttcaagt3241 tagataacaa aatgtttata cccattagaa cagagaataa atagaactac atttcttgcaSEQ ID NO: 12: Rhesus Serpina1 mRNA, transcript variant 6 (XM_001099255.2)   1 gcccagtctt gtgtctgcct ggcaatgggc aaggcccctt cctgcccaag ctccccgccc  61 ctccccaacc tattgcctcc gccacccgcc acccgaggcc aacttcctgg gtgggcagga 121 actgggccct gtgcccaggg cgtgcactgc ctccacgcag caaccctcag agtactgagc 181 tgagcaaagg aggaggaggg gatcagcact ctgaggaagg cctagccact gctgctgcca 241 ggaattccag ggcggcatca gtcttcagca tcaggcattt cggggtgaat tagtaaatgg 301 tagatcttgc taccagtgga acagccgcta aggattctgc agtgagagca gagggccagc 361 aaagtggtac tctcccagcg actggctgac tcacgccacc ccctccacct tggacgcagg 421 acactgtggt ttctgagcca ggtacaatga ctccttttgg tacgtgcagt ggaggctgta 481 tgctgctcag gcagagcgtc cggacagcgt gggcgggcga ctcagcgccc agcctgtgaa 541 cttagtccct gtttgctcct ccggtaactg gggtgatctt ggttaatatt caccagcagc 601 ctcccccgtt gcccctctgc acccactgct taaatacgga caaggacagg gctctgtctc 661 ctcagcctca ggcaccacca ctgacctggg acggtgaatc gacaatgcca tcttctgtct 721 catggggcgt cctcctgctg gcaggcctgt gctgcctgct ccccggctct ctggctgagg 781 atccccaggg agatgctgcc cagaagacgg atacatccca ccatgatcag gaccacccaa 841 ccctcaacaa gatcaccccc agcctggctg agttcggctt cagcctatac cgccagctgg 901 cacaccagtc caacagcacc aatatcttct tctccccagt gagcatcgct acagcctttg 961 caatgctctc cctggggacc aaggctgaca ctcacagtga aatcctggag ggcctgaatt1021 tcaacgtcac ggagattccg gaggctcagg tccatgaagg cttccaggaa ctcctccata1081 ccctcaacaa gccagacagc cagctccagc tgaccaccgg caacggcctg ttcctcaaca1141 agagcctgaa ggtagtggat aagtttttgg aggatgtcaa aaaactgtac cactcagaag1201 ccttctctgt caactttgag gacaccgaag aggccaagaa acagatcaac aattacgtgg1261 agaaggaaac tcaagggaaa attgtggatt tggtcaagga gcttgacaga gacacagttt1321 ttgctctggt gaattacatc ttctttaaag gcaaatggga gagacccttt gacgttgagg1381 ccaccaagga agaggacttc cacgtggacc aggcgaccac cgtgaaggtg cccatgatga1441 ggcgtttagg catgtttaac atctaccact gtgagaagct gtccagctgg gtgctgctga1501 tgaaatacct gggcaatgcc accgccatct tcttcctgcc tgatgagggg aaactgcagc1561 acctggaaaa tgaactcacc catgatatca tcaccaagtt cctggaaaat gaaaacagca1621 ggtctgccaa cttacattta cccagactgg ccattactgg aacctatgat ctgaagacag1681 tcctgggcca cctgggtatc actaaggtct tcagcaatgg ggctgacctc tcggggatca1741 cggaggaggc acccctgaag ctctccaagg ccgtgcataa ggctgtgctg accatcgatg1801 agaaagggac tgaagctgct ggggccatgt ttttagaggc catacccatg tctattcccc1861 ccgaggtcaa gttcaacaaa ccctttgtct tcttaatgat tgaacaaaat accaagtctc1921 ccctcttcat gggaaaagtg gtgaatccca cccagaaata actgcctgtc actcctcagc1981 ccctcccctc catccctggc cccctccctg aatgacatta aagaagggtt gagctggtcc2041 ctgcctgcgt gtgtgactgc aaacSEQ ID NO: 13: Rhesus Serpina1 mRNA, transcript variant 4 (XM_001099044.2)   1 tcttgtgtct gcctggcaat gggcaaggcc ccttcctgcc caagctcccc gcccctcccc  61 aacctattgc ctccgccacc cgccacccga ggccaacttc ctgggtgggc aggaactggg 121 ccctgtgccc agggcgtgca ctgcctccac gcagcaaccc tcagagtact gagctgagca 181 aaggaggagg aggggatcag cactctgagg aaggcctagc cactgctgct gccaggaatt 241 ccaggacaat gccatcttct gtctcatggg gcgtcctcct gctggcaggc ctgtgctgcc 301 tgctccccgg ctctctggct gaggatcccc agggagatgc tgcccagaag acggatacat 361 cccaccatga tcaggaccac ccaaccctca acaagatcac ccccagcctg gctgagttcg 421 gcttcagcct ataccgccag ctggcacacc agtccaacag caccaatatc ttcttctccc 481 cagtgagcat cgctacagcc tttgcaatgc tctccctggg gaccaaggct gacactcaca 541 gtgaaatcct ggagggcctg aatttcaacg tcacggagat tccggaggct caggtccatg 601 aaggcttcca ggaactcctc cataccctca acaagccaga cagccagctc cagctgacca 661 ccggcaacgg cctgttcctc aacaagagcc tgaaggtagt ggataagttt ttggaggatg 721 tcaaaaaact gtaccactca gaagccttct ctgtcaactt tgaggacacc gaagaggcca 781 agaaacagat caacaattac gtggagaagg aaactcaagg gaaaattgtg gatttggtca 841 aggagcttga cagagacaca gtttttgctc tggtgaatta catcttcttt aaaggcaaat 901 gggagagacc ctttgacgtt gaggccacca aggaagagga cttccacgtg gaccaggcga 961 ccaccgtgaa ggtgcccatg atgaggcgtt taggcatgtt taacatctac cactgtgaga1021 agctgtccag ctgggtgctg ctgatgaaat acctgggcaa tgccaccgcc atcttcttcc1081 tgcctgatga ggggaaactg cagcacctgg aaaatgaact cacccatgat atcatcacca1141 agttcctgga aaatgaaaac agcaggtctg ccaacttaca tttacccaga ctggccatta1201 ctggaaccta tgatctgaag acagtcctgg gccacctggg tatcactaag gtcttcagca1261 atggggctga cctctcgggg atcacggagg aggcacccct gaagctctcc aaggccgtgc1321 ataaggctgt gctgaccatc gatgagaaag ggactgaagc tgctggggcc atgtttttag1381 aggccatacc catgtctatt ccccccgagg tcaagttcaa caaacccttt gtcttcttaa1441 tgattgaaca aaataccaag tctcccctct tcatgggaaa agtggtgaat cccacccaga1501 aataactgcc tgtcactcct cagcccctcc cctccatccc tggccccctc cctgaatgac1561 attaaagaag ggttgagctg gtccctgcct gcgtgtgtga ctgcaaacSEQ ID NO: 14: Reverse complement of SEQ ID NO: 01tgcaagaaatgtagttctatttattctctgttctaatgggtataaacattttgttatctaacttgaacatcataccaactcaaaggcaggtcttactagcaatgactggggctcagagaggtttggcgacttcacgaaggtcacacagctgtcagggggaaaagtcagaacttggatccaggtcttcagactctcaggtctggtgtcatcctagggggcttggtgatggccatatctttaatgtatttgtggagagtgaaaggctgtcagtgagtaagcttaagagaacaggagacttgtgtgggaaacagtcggtatccattgattagactgaatcatgtagaattgctaatttcaccattttgaactatcgaaatcactatttggtatgactcaacctcatcctttaagtacacattcatgacagtgagtggttagacagtgattcctagattagtttgggatggggcagtgccttccataggacaaggccattcctggtagagacggagggagcaggctgtccttcagctaggggcccaggggacttcctggctgctgtgtcctttagcagggccttggaggatggaaggactctcctggcccttaccacaggggctattcaggaacagcctcctgccgtggcactggagctgcggtggccctctgaagactgcagggacagcaacaggcacaaagaagtcaggctgcatgtggccccagtcgggactcagaggaggaaagggaggggttgcgggggtcaatgggtgatgtgcttcctctcccatagctgaggagtccttgcaatggccttcctgagccatcagcaggcctatggccatgtgactagggaggagaagggatatagggtaatggtcttctggggcctgctgggcctggtcaagtctgaggaggagatagagaaaacaaagcagagaccctcctcttcatctggggagaagggacctgattctaaacggagatatgtgaggctttctggggcagcgatggaaggacaaggacagaataggtgtccttgttgccccatggagaatgggcttcaggaagaatctgcctcagtcattttccagaagtgcctgagaggtgcagggcccgagtctggttaggtgacagcgggtcaagaggaggacattgtcctctctcttgtgttgctgcagatgccacaaaactgggccacggcacgatcaggcagttctggggcccccaggagggcagccttggggtgggcacagccagccctggcaggatgagcaactctgggtgggggggagtgggggatgagcagggggacatgaagatgcttggtggagcctggggtcatggctggtatctggttcctcccctgtgattccttcttggggactccaagacaggacaaggaagactggagccctccagaaacagatgggcccaggtccgtaagctgaggattcagtcccccctggattcaagcccagcatgtgcctacccagccagatgctccatgaacacagttcagggggcccgaagacagcactgttacctggagcccacatacagcctcagcaggcaaagggagactcagagaaaacatgggagggatttacagtcacatgcaggcagggaccagctcaacccttctttaatgtcatccagggagggggccagggatggaggggaggggttgaggagcgagaggcagttatttttgggtgggattcaccacttttcccatgaagaggggagacttggtattttgttcaatcattaagaagacaaagggtttgttgaacttgacctcgggggggatagacatgggtatggcctctaaaaacatggccccagcagcttcagtccctttctcgtcgatggtcagcacagccttatgcacggccttggagagcttcaggggtgcctcctctgtgaccccggagaggtcagccccattgctgaagaccttagtgatgcccagttgacccaggacgctcttcagatcataggttccagtaatggacagtttgggtaaatgtaagctggcagaccttctgtcttcattttccaggaacttggtgatgatatcgtgggtgagttcattttccaggtgctgtagtttcccctcatcaggcaggaagaagatggcggtggcattgcccaggtatttcatcagcagcacccagctggacagcttcttacagtgctggatgttaaacatgcctaaacgcttcatcataggcaccttcacggtggtcacctggtccacgtggaagtcctcttcctcggtgtccttgacttcaaagggtctctcccatttgcctttaaagaagatgtaattcaccagagcaaaaactgtgtctctgtcaagctccttgaccaaatccacaattttcccttgagtacccttctccacgtaatcgttgatctgtttcttggcctcttcggtgtccccgaagttgacagtgaaggcttctgagtggtacaactttttaacatcctccaaaaacttatccactagcttcaggccctcgctgaggaacaggccattgccggtggtcagctggagctggctgtctggctggttgagggtacggaggagttcctggaagccttcatggatctgagcctccggaatctccgtgaggttgaaattcaggccctccaggatttcatcgtgagtgtcagccttggtccccagggagagcattgcaaaggctgtagcgatgctcactggggagaagaagatattggtgctgttggactggtgtgccagctggcggtataggctgaaggcgaactcagccaggttgggggtgatcttgttgaaggttgggtgatcctgatcatggtgggatgtatctgtcttctgggcagcatctccctggggatcctcagccagggagacagggaccaggcagcacaggcctgccagcaggaggatgccccacgagacagaagacggcattgtcgattcactgtcccaggtcagtggtggtgcctgaagctgaggagacagggccctgtcctcgtccgtatttaagcagtggatccagaggggcaacgggggaggctgctggtgaatattaaccaaggtcaccccagttatcggaggagcaaacaggggctaagtccactggctgggatctgagtcgcccgcctacgctgcccggacgctttgcctgggcagtgtacagcttccactgcacttaccgaaaggagtcattgtSEQ ID NO: 15: Reverse complement of SEQ ID NO: 02tgcaagaaatgtagttctatttattctctgttctaatgggtataaacattttgttatctaacttgaacatcataccaactcaaaggcaggtcttactagcaatgactggggctcagagaggtttggcgacttcacgaaggtcacacagctgtcagggggaaaagtcagaacttggatccaggtcttcagactctcaggtctggtgtcatcctagggggcttggtgatggccatatctttaatgtatttgtggagagtgaaaggctgtcagtgagtaagcttaagagaacaggagacttgtgtgggaaacagtcggtatccattgattagactgaatcatgtagaattgctaatttcaccattttgaactatcgaaatcactatttggtatgactcaacctcatcctttaagtacacattcatgacagtgagtggttagacagtgattcctagattagtttgggatggggcagtgccttccataggacaaggccattcctggtagagacggagggagcaggctgtccttcagctaggggcccaggggacttcctggctgctgtgtcctttagcagggccttggaggatggaaggactctcctggcccttaccacaggggctattcaggaacagcctcctgccgtggcactggagctgcggtggccctctgaagactgcagggacagcaacaggcacaaagaagtcaggctgcatgtggccccagtcgggactcagaggaggaaagggaggggttgcgggggtcaatgggtgatgtgcttcctctcccatagctgaggagtccttgcaatggccttcctgagccatcagcaggcctatggccatgtgactagggaggagaagggatatagggtaatggtcttctggggcctgctgggcctggtcaagtctgaggaggagatagagaaaacaaagcagagaccctcctcttcatctggggagaagggacctgattctaaacggagatatgtgaggctttctggggcagcgatggaaggacaaggacagaataggtgtccttgttgccccatggagaatgggcttcaggaagaatctgcctcagtcattttccagaagtgcctgagaggtgcagggcccgagtctggttaggtgacagcgggtcaagaggaggacattgtcctctctcttgtgttgctgcagatgccacaaaactgggccacggcacgatcaggcagttctggggcccccaggagggcagccttggggtgggcacagccagccctggcaggatgagcaactctgggtgggggggagtgggggatgagcagggggacatgaagatgcttggtggagcctggggtcatggctggtatctggttcctcccctgtgattccttcttggggactccaagacaggacaaggaagactggagccctccagaaacagatgggcccaggtccgtaagctgaggattcagtcccccctggattcaagcccagcatgtgcctacccagccagatgctccatgaacacagttcagggggcccgaagacagcactgttacctggagcccacatacagcctcagcaggcaaagggagactcagagaaaacatgggagggatttacagtcacatgcaggcagggaccagctcaacccttctttaatgtcatccagggagggggccagggatggaggggaggggttgaggagcgagaggcagttatttttgggtgggattcaccacttttcccatgaagaggggagacttggtattttgttcaatcattaagaagacaaagggtttgttgaacttgacctcgggggggatagacatgggtatggcctctaaaaacatggccccagcagcttcagtccctttctcgtcgatggtcagcacagccttatgcacggccttggagagcttcaggggtgcctcctctgtgaccccggagaggtcagccccattgctgaagaccttagtgatgcccagttgacccaggacgctcttcagatcataggttccagtaatggacagtttgggtaaatgtaagctggcagaccttctgtcttcattttccaggaacttggtgatgatatcgtgggtgagttcattttccaggtgctgtagtttcccctcatcaggcaggaagaagatggcggtggcattgcccaggtatttcatcagcagcacccagctggacagcttcttacagtgctggatgttaaacatgcctaaacgcttcatcataggcaccttcacggtggtcacctggtccacgtggaagtcctcttcctcggtgtccttgacttcaaagggtctctcccatttgcctttaaagaagatgtaattcaccagagcaaaaactgtgtctctgtcaagctccttgaccaaatccacaattttcccttgagtacccttctccacgtaatcgttgatctgtttcttggcctcttcggtgtccccgaagttgacagtgaaggcttctgagtggtacaactttttaacatcctccaaaaacttatccactagcttcaggccctcgctgaggaacaggccattgccggtggtcagctggagctggctgtctggctggttgagggtacggaggagttcctggaagccttcatggatctgagcctccggaatctccgtgaggttgaaattcaggccctccaggatttcatcgtgagtgtcagccttggtccccagggagagcattgcaaaggctgtagcgatgctcactggggagaagaagatattggtgctgttggactggtgtgccagctggcggtataggctgaaggcgaactcagccaggttgggggtgatcttgttgaaggttgggtgatcctgatcatggtgggatgtatctgtcttctgggcagcatctccctggggatcctcagccagggagacagggaccaggcagcacaggcctgccagcaggaggatgccccacgagacagaagacggcattgtcctttcctgctctcctcaagctctcctcaagctctgtctcttctggcaggcacaggagagtggcctgaaggctggcaggaggttgccgcccctccaacctggaattcctggcagcagcagcggctaggccttcctcggaggcccgaccccctcctccttcttggttcagctcaggactctgagggttgctgcgtggaggcagtgcatgccctgggcacagtgcccagttcctgcccaSEQ ID NO: 16: Reverse complement of SEQ ID NO: 03tgcaagaaatgtagttctatttattctctgttctaatgggtataaacattttgttatctaacttgaacatcataccaactcaaaggcaggtcttactagcaatgactggggctcagagaggtttggcgacttcacgaaggtcacacagctgtcagggggaaaagtcagaacttggatccaggtcttcagactctcaggtctggtgtcatcctagggggcttggtgatggccatatctttaatgtatttgtggagagtgaaaggctgtcagtgagtaagcttaagagaacaggagacttgtgtgggaaacagtcggtatccattgattagactgaatcatgtagaattgctaatttcaccattttgaactatcgaaatcactatttggtatgactcaacctcatcctttaagtacacattcatgacagtgagtggttagacagtgattcctagattagtttgggatggggcagtgccttccataggacaaggccattcctggtagagacggagggagcaggctgtccttcagctaggggcccaggggacttcctggctgctgtgtcctttagcagggccttggaggatggaaggactctcctggcccttaccacaggggctattcaggaacagcctcctgccgtggcactggagctgcggtggccctctgaagactgcagggacagcaacaggcacaaagaagtcaggctgcatgtggccccagtcgggactcagaggaggaaagggaggggttgcgggggtcaatgggtgatgtgcttcctctcccatagctgaggagtccttgcaatggccttcctgagccatcagcaggcctatggccatgtgactagggaggagaagggatatagggtaatggtcttctggggcctgctgggcctggtcaagtctgaggaggagatagagaaaacaaagcagagaccctcctcttcatctggggagaagggacctgattctaaacggagatatgtgaggctttctggggcagcgatggaaggacaaggacagaataggtgtccttgttgccccatggagaatgggcttcaggaagaatctgcctcagtcattttccagaagtgcctgagaggtgcagggcccgagtctggttaggtgacagcgggtcaagaggaggacattgtcctctctcttgtgttgctgcagatgccacaaaactgggccacggcacgatcaggcagttctggggcccccaggagggcagccttggggtgggcacagccagccctggcaggatgagcaactctgggtgggggggagtgggggatgagcagggggacatgaagatgcttggtggagcctggggtcatggctggtatctggttcctcccctgtgattccttcttggggactccaagacaggacaaggaagactggagccctccagaaacagatgggcccaggtccgtaagctgaggattcagtcccccctggattcaagcccagcatgtgcctacccagccagatgctccatgaacacagttcagggggcccgaagacagcactgttacctggagcccacatacagcctcagcaggcaaagggagactcagagaaaacatgggagggatttacagtcacatgcaggcagggaccagctcaacccttctttaatgtcatccagggagggggccagggatggaggggaggggttgaggagcgagaggcagttatttttgggtgggattcaccacttttcccatgaagaggggagacttggtattttgttcaatcattaagaagacaaagggtttgttgaacttgacctcgggggggatagacatgggtatggcctctaaaaacatggccccagcagcttcagtccctttctcgtcgatggtcagcacagccttatgcacggccttggagagcttcaggggtgcctcctctgtgaccccggagaggtcagccccattgctgaagaccttagtgatgcccagttgacccaggacgctcttcagatcataggttccagtaatggacagtttgggtaaatgtaagctggcagaccttctgtcttcattttccaggaacttggtgatgatatcgtgggtgagttcattttccaggtgctgtagtttcccctcatcaggcaggaagaagatggcggtggcattgcccaggtatttcatcagcagcacccagctggacagcttcttacagtgctggatgttaaacatgcctaaacgcttcatcataggcaccttcacggtggtcacctggtccacgtggaagtcctcttcctcggtgtccttgacttcaaagggtctctcccatttgcctttaaagaagatgtaattcaccagagcaaaaactgtgtctctgtcaagctccttgaccaaatccacaattttcccttgagtacccttctccacgtaatcgttgatctgtttcttggcctcttcggtgtccccgaagttgacagtgaaggcttctgagtggtacaactttttaacatcctccaaaaacttatccactagcttcaggccctcgctgaggaacaggccattgccggtggtcagctggagctggctgtctggctggttgagggtacggaggagttcctggaagccttcatggatctgagcctccggaatctccgtgaggttgaaattcaggccctccaggatttcatcgtgagtgtcagccttggtccccagggagagcattgcaaaggctgtagcgatgctcactggggagaagaagatattggtgctgttggactggtgtgccagctggcggtataggctgaaggcgaactcagccaggttgggggtgatcttgttgaaggttgggtgatcctgatcatggtgggatgtatctgtcttctgggcagcatctccctggggatcctcagccagggagacagggaccaggcagcacaggcctgccagcaggaggatgccccacgagacagaagacggcattgtcgattcactgtcccaggtcagtggtggtgcctgaagctgaggagacagggccctgtcctcgtccgtatttaagcagtggatccagaggggcaacgggggaggctgcgaaaggagtcattgtacctggctcagaaaccacagcgtcctgtgtccaaggtggagggggtggcgtgagtcagacagtctctgggagagtaccacttagctggccctctgctctcactgcagaatccttagtggctgttccactggtagcaagatctaccatttactgagtcaccccaaaatgcctgatgctgaagacttactgccgccctttcctgctctcctcaagctctcctcaagctctgtctcttctggcaggcacaggagagtggcctgaaggctggcaggaggttgccgcccctccaacctggaattcctggcagcagcagcggctaggccttcctcggaggcccgaccccctcctccttcttggttcagctcaggactctgagggttgctgcgtggaggcagtgcatgccctgggcacagtgcccagttcctgcccaSEQ ID NO: 17: Reverse complement of SEQ ID NO: 04tgcaagaaatgtagttctatttattctctgttctaatgggtataaacattttgttatctaacttgaacatcataccaactcaaaggcaggtcttactagcaatgactggggctcagagaggtttggcgacttcacgaaggtcacacagctgtcagggggaaaagtcagaacttggatccaggtcttcagactctcaggtctggtgtcatcctagggggcttggtgatggccatatctttaatgtatttgtggagagtgaaaggctgtcagtgagtaagcttaagagaacaggagacttgtgtgggaaacagtcggtatccattgattagactgaatcatgtagaattgctaatttcaccattttgaactatcgaaatcactatttggtatgactcaacctcatcctttaagtacacattcatgacagtgagtggttagacagtgattcctagattagtttgggatggggcagtgccttccataggacaaggccattcctggtagagacggagggagcaggctgtccttcagctaggggcccaggggacttcctggctgctgtgtcctttagcagggccttggaggatggaaggactctcctggccataccacaggggctattcaggaacagcctcctgccgtggcactggagctgcggtggccctctgaagactgcagggacagcaacaggcacaaagaagtcaggctgcatgtggccccagtcgggactcagaggaggaaagggaggggttgcgggggtcaatgggtgatgtgcttcctctcccatagctgaggagtccttgcaatggccttcctgagccatcagcaggcctatggccatgtgactagggaggagaagggatatagggtaatggtcttctggggcctgctgggcctggtcaagtctgaggaggagatagagaaaacaaagcagagaccctcctatcatctggggagaagggacctgattctaaacggagatatgtgaggctttctggggcagcgatggaaggacaaggacagaataggtgtccttgttgccccatggagaatgggcttcaggaagaatctgcctcagtcattttccagaagtgcctgagaggtgcagggcccgagtctggttaggtgacagcgggtcaagaggaggacattgtcctctctcttgtgttgctgcagatgccacaaaactgggccacggcacgatcaggcagttctggggcccccaggagggcagccttggggtgggcacagccagccctggcaggatgagcaactctgggtgggggggagtgggggatgagcagggggacatgaagatgcttggtggagcctggggtcatggctggtatctggttcctcccctgtgattccttcttggggactccaagacaggacaaggaagactggagccctccagaaacagatgggcccaggtccgtaagctgaggattcagtcccccctggattcaagcccagcatgtgcctacccagccagatgctccatgaacacagttcagggggcccgaagacagcactgttacctggagcccacatacagcctcagcaggcaaagggagactcagagaaaacatgggagggatttacagtcacatgcaggcagggaccagctcaacccttctttaatgtcatccagggagggggccagggatggaggggaggggttgaggagcgagaggcagttatttttgggtgggattcaccacttttcccatgaagaggggagacttggtattttgttcaatcattaagaagacaaagggtttgttgaacttgacctcgggggggatagacatgggtatggcctctaaaaacatggccccagcagcttcagtccctttctcgtcgatggtcagcacagccttatgcacggccttggagagcttcaggggtgcctcctctgtgaccccggagaggtcagccccattgctgaagaccttagtgatgcccagttgacccaggacgctcttcagatcataggttccagtaatggacagtttgggtaaatgtaagctggcagaccttctgtcttcattttccaggaacttggtgatgatatcgtgggtgagttcattttccaggtgctgtagtttcccctcatcaggcaggaagaagatggcggtggcattgcccaggtatttcatcagcagcacccagctggacagcttcttacagtgctggatgttaaacatgcctaaacgcttcatcataggcaccttcacggtggtcacctggtccacgtggaagtcctcttcctcggtgtccttgacttcaaagggtctctcccatttgcctttaaagaagatgtaattcaccagagcaaaaactgtgtctctgtcaagctccttgaccaaatccacaattttcccttgagtacccttctccacgtaatcgttgatctgtttcttggcctcttcggtgtccccgaagttgacagtgaaggcttctgagtggtacaactttttaacatcctccaaaaacttatccactagcttcaggccctcgctgaggaacaggccattgccggtggtcagctggagctggctgtctggctggttgagggtacggaggagttcctggaagccttcatggatctgagcctccggaatctccgtgaggttgaaattcaggccctccaggatttcatcgtgagtgtcagccttggtccccagggagagcattgcaaaggctgtagcgatgctcactggggagaagaagatattggtgctgttggactggtgtgccagctggcggtataggctgaaggcgaactcagccaggttgggggtgatcttgttgaaggttgggtgatcctgatcatggtgggatgtatctgtcttctgggcagcatctccctggggatcctcagccagggagacagggaccaggcagcacaggcctgccagcaggaggatgccccacgagacagaagacggcattgtcctgtggaactgagtgagcagcagcagcaatgtcccacctttcctgctctcctcaagctctcctcaagctctgtctcttctggcaggcacaggagagtggcctgaaggctggcaggaggttgccgcccctccaacctggaattcctggcagcagcagcggctaggccttcctcggaggcccgaccccctcctccttcttggttcagctcaggactctgagggttgctgcgtggaggcagtgcatgccctgggcacagtgcccagttcctgcccaSEQ ID NO: 18: Reverse complement of SEQ ID NO: 05tgcaagaaatgtagttctatttattctctgttctaatgggtataaacattttgttatctaacttgaacatcataccaactcaaaggcaggtcttactagcaatgactggggctcagagaggtttggcgacttcacgaaggtcacacagctgtcagggggaaaagtcagaacttggatccaggtcttcagactctcaggtctggtgtcatcctagggggcttggtgatggccatatctttaatgtatttgtggagagtgaaaggctgtcagtgagtaagcttaagagaacaggagacttgtgtgggaaacagtcggtatccattgattagactgaatcatgtagaattgctaatttcaccattttgaactatcgaaatcactatttggtatgactcaacctcatcctttaagtacacattcatgacagtgagtggttagacagtgattcctagattagtttgggatggggcagtgccttccataggacaaggccattcctggtagagacggagggagcaggctgtccttcagctaggggcccaggggacttcctggctgctgtgtcctttagcagggccttggaggatggaaggactctcctggcccttaccacaggggctattcaggaacagcctcctgccgtggcactggagctgcggtggccctctgaagactgcagggacagcaacaggcacaaagaagtcaggctgcatgtggccccagtcgggactcagaggaggaaagggaggggttgcgggggtcaatgggtgatgtgcttcctctcccatagctgaggagtccttgcaatggccttcctgagccatcagcaggcctatggccatgtgactagggaggagaagggatatagggtaatggtcttctggggcctgctgggcctggtcaagtctgaggaggagatagagaaaacaaagcagagaccctcctcttcatctggggagaagggacctgattctaaacggagatatgtgaggctttctggggcagcgatggaaggacaaggacagaataggtgtccttgttgccccatggagaatgggcttcaggaagaatctgcctcagtcattttccagaagtgcctgagaggtgcagggcccgagtctggttaggtgacagcgggtcaagaggaggacattgtcctctctcttgtgttgctgcagatgccacaaaactgggccacggcacgatcaggcagttctggggcccccaggagggcagccttggggtgggcacagccagccctggcaggatgagcaactctgggtgggggggagtgggggatgagcagggggacatgaagatgcttggtggagcctggggtcatggctggtatctggttcctcccctgtgattccttcttggggactccaagacaggacaaggaagactggagccctccagaaacagatgggcccaggtccgtaagctgaggattcagtcccccctggattcaagcccagcatgtgcctacccagccagatgctccatgaacacagttcagggggcccgaagacagcactgttacctggagcccacatacagcctcagcaggcaaagggagactcagagaaaacatgggagggatttacagtcacatgcaggcagggaccagctcaacccttctttaatgtcatccagggagggggccagggatggaggggaggggttgaggagcgagaggcagttatttttgggtgggattcaccacttttcccatgaagaggggagacttggtattttgttcaatcattaagaagacaaagggtttgttgaacttgacctcgggggggatagacatgggtatggcctctaaaaacatggccccagcagcttcagtccctttctcgtcgatggtcagcacagccttatgcacggccttggagagcttcaggggtgcctcctctgtgaccccggagaggtcagccccattgctgaagaccttagtgatgcccagttgacccaggacgctcttcagatcataggttccagtaatggacagtttgggtaaatgtaagctggcagaccttctgtcttcattttccaggaacttggtgatgatatcgtgggtgagttcattttccaggtgctgtagtttcccctcatcaggcaggaagaagatggcggtggcattgcccaggtatttcatcagcagcacccagctggacagcttcttacagtgctggatgttaaacatgcctaaacgcttcatcataggcaccttcacggtggtcacctggtccacgtggaagtcctcttcctcggtgtccttgacttcaaagggtctctcccatttgcctttaaagaagatgtaattcaccagagcaaaaactgtgtctctgtcaagctccttgaccaaatccacaattttcccttgagtacccttctccacgtaatcgttgatctgtttcttggcctcttcggtgtccccgaagttgacagtgaaggcttctgagtggtacaactttttaacatcctccaaaaacttatccactagcttcaggccctcgctgaggaacaggccattgccggtggtcagctggagctggctgtctggctggttgagggtacggaggagttcctggaagccttcatggatctgagcctccggaatctccgtgaggttgaaattcaggccctccaggatttcatcgtgagtgtcagccttggtccccagggagagcattgcaaaggctgtagcgatgctcactggggagaagaagatattggtgctgttggactggtgtgccagctggcggtataggctgaaggcgaactcagccaggttgggggtgatcttgttgaaggttgggtgatcctgatcatggtgggatgtatctgtcttctgggcagcatctccctggggatcctcagccagggagacagggaccaggcagcacaggcctgccagcaggaggatgccccacgagacagaagacggcattgtcgattcactgtcccaggtcagtggtggtgcctgaagctgaggagacagggccctgtcctcgtccgtatttaagcagtggatccagaggggcaacgggggaggctgctggctcagaaaccacagcgtcctgtgtccaaggtggagggggtggcgtgagtcagacagtctctgggagagtaccacttagctggccctctgctctcactgcagaatccttagtggctgttccactggtagcaagatctaccatttactgagtcaccccaaaatgcctgatgctgaagacttactgccgccctgtggaactgagtgagcagcagcagcaatgtcccacctttcctgctctcctcaagctctcctcaagctctgtctcttctggcaggcacaggagagtggcctgaaggctggcaggaggttgccgcccctccaacctggaattcctggcagcagcagcggctaggccttcctcggaggcccgaccccctcctccttcttggttcagctcaggactctgagggttgctgcgtggaggcagtgcatgccctgggcacagtgcccagttcctgccca SEQ ID NO: 19: Reverse complement of SEQ ID NO: 06tgcaagaaatgtagttctatttattctctgttctaatgggtataaacattttgttatctaacttgaacatcataccaactcaaaggcaggtcttactagcaatgactggggctcagagaggtttggcgacttcacgaaggtcacacagctgtcagggggaaaagtcagaacttggatccaggtcttcagactctcaggtctggtgtcatcctagggggcttggtgatggccatatctttaatgtatttgtggagagtgaaaggctgtcagtgagtaagcttaagagaacaggagacttgtgtgggaaacagtcggtatccattgattagactgaatcatgtagaattgctaatttcaccattttgaactatcgaaatcactatttggtatgactcaacctcatcctttaagtacacattcatgacagtgagtggttagacagtgattcctagattagtttgggatggggcagtgccttccataggacaaggccattcctggtagagacggagggagcaggctgtccttcagctaggggcccaggggacttcctggctgctgtgtcctttagcagggccttggaggatggaaggactctcctggcccttaccacaggggctattcaggaacagcctcctgccgtggcactggagctgcggtggccctctgaagactgcagggacagcaacaggcacaaagaagtcaggctgcatgtggccccagtcgggactcagaggaggaaagggaggggttgcgggggtcaatgggtgatgtgcttcctctcccatagctgaggagtccttgcaatggccttcctgagccatcagcaggcctatggccatgtgactagggaggagaagggatatagggtaatggtcttctggggcctgctgggcctggtcaagtctgaggaggagatagagaaaacaaagcagagaccctcctcttcatctggggagaagggacctgattctaaacggagatatgtgaggctttctggggcagcgatggaaggacaaggacagaataggtgtccttgttgccccatggagaatgggcttcaggaagaatctgcctcagtcattttccagaagtgcctgagaggtgcagggcccgagtctggttaggtgacagcgggtcaagaggaggacattgtcctctctcttgtgttgctgcagatgccacaaaactgggccacggcacgatcaggcagttctggggcccccaggagggcagccttggggtgggcacagccagccctggcaggatgagcaactctgggtgggggggagtgggggatgagcagggggacatgaagatgcttggtggagcctggggtcatggctggtatctggttcctcccctgtgattccttcttggggactccaagacaggacaaggaagactggagccctccagaaacagatgggcccaggtccgtaagctgaggattcagtcccccctggattcaagcccagcatgtgcctacccagccagatgctccatgaacacagttcagggggcccgaagacagcactgttacctggagcccacatacagcctcagcaggcaaagggagactcagagaaaacatgggagggatttacagtcacatgcaggcagggaccagctcaacccttctttaatgtcatccagggagggggccagggatggaggggaggggttgaggagcgagaggcagttatttttgggtgggattcaccacttttcccatgaagaggggagacttggtattttgttcaatcattaagaagacaaagggtttgttgaacttgacctcgggggggatagacatgggtatggcctctaaaaacatggccccagcagcttcagtccctttctcgtcgatggtcagcacagccttatgcacggccttggagagcttcaggggtgcctcctctgtgaccccggagaggtcagccccattgctgaagaccttagtgatgcccagttgacccaggacgctcttcagatcataggttccagtaatggacagtttgggtaaatgtaagctggcagaccttctgtcttcattttccaggaacttggtgatgatatcgtgggtgagttcattttccaggtgctgtagtttcccctcatcaggcaggaagaagatggcggtggcattgcccaggtatttcatcagcagcacccagctggacagcttcttacagtgctggatgttaaacatgcctaaacgcttcatcataggcaccttcacggtggtcacctggtccacgtggaagtcctcttcctcggtgtccttgacttcaaagggtctctcccatttgcctttaaagaagatgtaattcaccagagcaaaaactgtgtctctgtcaagctccttgaccaaatccacaattttcccttgagtacccttctccacgtaatcgttgatctgtttcttggcctcttcggtgtccccgaagttgacagtgaaggcttctgagtggtacaactttttaacatcctccaaaaacttatccactagcttcaggccctcgctgaggaacaggccattgccggtggtcagctggagctggctgtctggctggttgagggtacggaggagttcctggaagccttcatggatctgagcctccggaatctccgtgaggttgaaattcaggccctccaggatttcatcgtgagtgtcagccttggtccccagggagagcattgcaaaggctgtagcgatgctcactggggagaagaagatattggtgctgttggactggtgtgccagctggcggtataggctgaaggcgaactcagccaggttgggggtgatcttgttgaaggttgggtgatcctgatcatggtgggatgtatctgtcttctgggcagcatctccctggggatcctcagccagggagacagggaccaggcagcacaggcctgccagcaggaggatgccccacgagacagaagacggcattgtcgattcactgtcccaggtcagtggtggtgcctgaagctgaggagacagggccctgtcctcgtccgtatttaagcagtggatccagaggggcaacgggggaggctgctgtggaactgagtgagcagcagcagcaatgtcccacctttcctgctctcctcaagctctcctcaagctctgtctcttctggcaggcacaggagagtggcctgaaggctggcaggaggttgccgcccctccaacctggaattcctggcagcagcagcggctaggccttcctcggaggcccgaccccctcctccttcttggttcagctcaggactctgagggttgctgcgtggaggcagtgcatgccctgggcacagtgcccagttcctgcccaSEQ ID NO: 20: Reverse complement of SEQ ID NO: 07tgcaagaaatgtagttctatttattctctgttctaatgggtataaacattttgttatctaacttgaacatcataccaactcaaaggcaggtatactagcaatgactggggctcagagaggtttggcgacttcacgaaggtcacacagctgtcagggggaaaagtcagaacttggatccaggtcttcagactctcaggtctggtgtcatcctagggggcttggtgatggccatatctttaatgtatttgtggagagtgaaaggctgtcagtgagtaagcttaagagaacaggagacttgtgtgggaaacagtcggtatccattgattagactgaatcatgtagaattgctaatttcaccattttgaactatcgaaatcactatttggtatgactcaacctcatcctttaagtacacattcatgacagtgagtggttagacagtgattcctagattagtttgggatggggcagtgccttccataggacaaggccattcctggtagagacggagggagcaggctgtccttcagctaggggcccaggggacttcctggctgctgtgtcctttagcagggccttggaggatggaaggactctcctggccdtaccacaggggctattcaggaacagcctcctgccgtggcactggagctgcggtggccctctgaagactgcagggacagcaacaggcacaaagaagtcaggctgcatgtggccccagtcgggactcagaggaggaaagggaggggttgcgggggtcaatgggtgatgtgcttcctctcccatagctgaggagtccttgcaatggccttcctgagccatcagcaggcctatggccatgtgactagggaggagaagggatatagggtaatggtcttctggggcctgctgggcctggtcaagtctgaggaggagatagagaaaacaaagcagagaccctcctcttcatctggggagaagggacctgattctaaacggagatatgtgaggctttctggggcagcgatggaaggacaaggacagaataggtgtccttgttgccccatggagaatgggcttcaggaagaatctgcctcagtcattttccagaagtgcctgagaggtgcagggcccgagtctggttaggtgacagcgggtcaagaggaggacattgtcctctctcttgtgttgctgcagatgccacaaaactgggccacggcacgatcaggcagttctggggcccccaggagggcagccttggggtgggcacagccagccctggcaggatgagcaactctgggtgggggggagtgggggatgagcagggggacatgaagatgcttggtggagcctggggtcatggctggtatctggttcctcccctgtgattccttcttggggactccaagacaggacaaggaagactggagccctccagaaacagatgggcccaggtccgtaagctgaggattcagtcccccctggattcaagcccagcatgtgcctacccagccagatgctccatgaacacagttcagggggcccgaagacagcactgttacctggagcccacatacagcctcagcaggcaaagggagactcagagaaaacatgggagggatttacagtcacatgcaggcagggaccagctcaacccttctttaatgtcatccagggagggggccagggatggaggggaggggttgaggagcgagaggcagttatttttgggtgggattcaccacttttcccatgaagaggggagacttggtattttgttcaatcattaagaagacaaagggtttgttgaacttgacctcgggggggatagacatgggtatggcctctaaaaacatggccccagcagcttcagtccctttctcgtcgatggtcagcacagccttatgcacggccttggagagcttcaggggtgcctcctctgtgaccccggagaggtcagccccattgctgaagaccttagtgatgcccagttgacccaggacgctcttcagatcataggttccagtaatggacagtttgggtaaatgtaagctggcagaccttctgtcttcattttccaggaacttggtgatgatatcgtgggtgagttcattttccaggtgctgtagtttcccctcatcaggcaggaagaagatggcggtggcattgcccaggtatttcatcagcagcacccagctggacagcttcttacagtgctggatgttaaacatgcctaaacgcttcatcataggcaccttcacggtggtcacctggtccacgtggaagtcctcttcctcggtgtccttgacttcaaagggtctctcccatttgcctttaaagaagatgtaattcaccagagcaaaaactgtgtctctgtcaagctccttgaccaaatccacaattttcccttgagtacccttctccacgtaatcgttgatctgtttcttggcctcttcggtgtccccgaagttgacagtgaaggcttctgagtggtacaactttttaacatcctccaaaaacttatccactagcttcaggccctcgctgaggaacaggccattgccggtggtcagctggagctggctgtctggctggttgagggtacggaggagttcctggaagccttcatggatctgagcctccggaatctccgtgaggttgaaattcaggccctccaggatttcatcgtgagtgtcagccttggtccccagggagagcattgcaaaggctgtagcgatgctcactggggagaagaagatattggtgctgttggactggtgtgccagctggcggtataggctgaaggcgaactcagccaggttgggggtgatcttgttgaaggttgggtgatcctgatcatggtgggatgtatctgtcttctgggcagcatctccctggggatcctcagccagggagacagggaccaggcagcacaggcctgccagcaggaggatgccccacgagacagaagacggcattgtcgattcactgtcccaggtcagtggtggtgcctgaagctgaggagacagggccctgtcctcgtccgtatttaagcagtggatccagaggggcaacgggggaggctgctggctcagaaaccacagcgtcctgtgtccaaggtggagggggtggcgtgagtcagacagtctctgggagagtaccacttagctggccctctgctctcactgcagaatccttagtggctgttccactggtagcaagatctaccatttactgagtcaccccaaaatgcctgatgctgaagacttactgccgccctttcctgctctcctcaagctctcctcaagctctgtctcttctggcaggcacaggagagtggcctgaaggctggcaggaggttgccgcccctccaacctggaattcctggcagcagcagcggctaggccttcctcggaggcccgaccccctcctccttcttggttcagctcaggactctgagggttgctgcgtggaggcagtgcatgccctgggcacagtgcccagttcctgcccaSEQ ID NO: 21: Reverse complement of SEQ ID NO: 08tgcaagaaatgtagttctatttattctctgttctaatgggtataaacattttgttatctaacttgaacatcataccaactcaaaggcaggtcttactagcaatgactggggctcagagaggtttggcgacttcacgaaggtcacacagctgtcagggggaaaagtcagaacttggatccaggtcttcagactctcaggtctggtgtcatcctagggggcttggtgatggccatatctttaatgtatttgtggagagtgaaaggctgtcagtgagtaagcttaagagaacaggagacttgtgtgggaaacagtcggtatccattgattagactgaatcatgtagaattgctaatttcaccattttgaactatcgaaatcactatttggtatgactcaacctcatcctttaagtacacattcatgacagtgagtggttagacagtgattcctagattagtttgggatggggcagtgccttccataggacaaggccattcctggtagagacggagggagcaggctgtccttcagctaggggcccaggggacttcctggctgctgtgtcctttagcagggccttggaggatggaaggactctcctggcccttaccacaggggctattcaggaacagcctcctgccgtggcactggagctgcggtggccctctgaagactgcagggacagcaacaggcacaaagaagtcaggctgcatgtggccccagtcgggactcagaggaggaaagggaggggttgcgggggtcaatgggtgatgtgcttcctctcccatagctgaggagtccttgcaatggccttcctgagccatcagcaggcctatggccatgtgactagggaggagaagggatatagggtaatggtcttctggggcctgctgggcctggtcaagtctgaggaggagatagagaaaacaaagcagagaccctcctcttcatctggggagaagggacctgattctaaacggagatatgtgaggctttctggggcagcgatggaaggacaaggacagaataggtgtccttgttgccccatggagaatgggcttcaggaagaatctgcctcagtcattttccagaagtgcctgagaggtgcagggcccgagtctggttaggtgacagcgggtcaagaggaggacattgtcctctctcttgtgttgctgcagatgccacaaaactgggccacggcacgatcaggcagttctggggcccccaggagggcagccttggggtgggcacagccagccctggcaggatgagcaactctgggtgggggggagtgggggatgagcagggggacatgaagatgcttggtggagcctggggtcatggctggtatctggttcctcccctgtgattccttcttggggactccaagacaggacaaggaagactggagccctccagaaacagatgggcccaggtccgtaagctgaggattcagtcccccctggattcaagcccagcatgtgcctacccagccagatgctccatgaacacagttcagggggcccgaagacagcactgttacctggagcccacatacagcctcagcaggcaaagggagactcagagaaaacatgggagggatttacagtcacatgcaggcagggaccagctcaacccttctttaatgtcatccagggagggggccagggatggaggggaggggttgaggagcgagaggcagttatttttgggtgggattcaccacttttcccatgaagaggggagacttggtattttgttcaatcattaagaagacaaagggtttgttgaacttgacctcgggggggatagacatgggtatggcctctaaaaacatggccccagcagcttcagtccctttctcgtcgatggtcagcacagccttatgcacggccttggagagcttcaggggtgcctcctctgtgaccccggagaggtcagccccattgctgaagaccttagtgatgcccagttgacccaggacgctcttcagatcataggttccagtaatggacagtttgggtaaatgtaagctggcagaccttctgtcttcattttccaggaacttggtgatgatatcgtgggtgagttcattttccaggtgctgtagtttcccctcatcaggcaggaagaagatggcggtggcattgcccaggtatttcatcagcagcacccagctggacagcttcttacagtgctggatgttaaacatgcctaaacgcttcatcataggcaccttcacggtggtcacctggtccacgtggaagtcctcttcctcggtgtccttgacttcaaagggtctctcccatttgcctttaaagaagatgtaattcaccagagcaaaaactgtgtctctgtcaagctccttgaccaaatccacaattttcccttgagtacccttctccacgtaatcgttgatctgtttcttggcctcttcggtgtccccgaagttgacagtgaaggcttctgagtggtacaactttttaacatcctccaaaaacttatccactagcttcaggccctcgctgaggaacaggccattgccggtggtcagctggagctggctgtctggctggttgagggtacggaggagttcctggaagccttcatggatctgagcctccggaatctccgtgaggttgaaattcaggccctccaggatttcatcgtgagtgtcagccttggtccccagggagagcattgcaaaggctgtagcgatgctcactggggagaagaagatattggtgctgttggactggtgtgccagctggcggtataggctgaaggcgaactcagccaggttgggggtgatcttgttgaaggttgggtgatcctgatcatggtgggatgtatctgtcttctgggcagcatctccctggggatcctcagccagggagacagggaccaggcagcacaggcctgccagcaggaggatgccccacgagacagaagacggcattgtcgattcactgtcccaggtcagtggtggtgcctgaagctgaggagacagggccctgtcctcgtccgtatttaagcagtggatccagaggggcaacgggggaggctggctcagaaaccacagcgtcctgtgtccaaggtggagggggtggcgtgagtcagacagtctctgggagagtaccacttagctggccctctgctctcactgcagaatccttagtggctgttccactggtagcaagatctaccatttactgagtcaccccaaaatgcctgatgctgaagacttactgccgccctttcctgctctcctcaagctctcctcaagctctgtctcttctggcaggcacaggagagtggcctgaaggctggcaggaggttgccgcccctccaacctggaattcctggcagcagcagcggctaggccttcctcggaggcccgaccccctcctccttcttggttcagctcaggactctgagggttgctgcgtggaggcagtgcatgccctgggcacagtgcccagttcctgcccaSEQ ID NO: 22: Reverse complement of SEQ ID NO: 09tgcaagaaatgtagttctatttattctctgttctaatgggtataaacattttgttatctaacttgaacatcataccaactcaaaggcaggtcttactagcaatgactggggctcagagaggtttggcgacttcacgaaggtcacacagctgtcagggggaaaagtcagaacttggatccaggtcttcagactctcaggtctggtgtcatcctagggggcttggtgatggccatatctttaatgtatttgtggagagtgaaaggctgtcagtgagtaagcttaagagaacaggagacttgtgtgggaaacagtcggtatccattgattagactgaatcatgtagaattgctaatttcaccattttgaactatcgaaatcactatttggtatgactcaacctcatcctttaagtacacattcatgacagtgagtggttagacagtgattcctagattagtttgggatggggcagtgccttccataggacaaggccattcctggtagagacggagggagcaggctgtccttcagctaggggcccaggggacttcctggctgctgtgtcctttagcagggccttggaggatggaaggactctcctggcccttaccacaggggctattcaggaacagcctcctgccgtggcactggagctgcggtggccctctgaagactgcagggacagcaacaggcacaaagaagtcaggctgcatgtggccccagtcgggactcagaggaggaaagggaggggttgcgggggtcaatgggtgatgtgcttcctctcccatagctgaggagtccttgcaatggccttcctgagccatcagcaggcctatggccatgtgactagggaggagaagggatatagggtaatggtcttctggggcctgctgggcctggtcaagtctgaggaggagatagagaaaacaaagcagagaccctcctcttcatctggggagaagggacctgattctaaacggagatatgtgaggctttctggggcagcgatggaaggacaaggacagaataggtgtccttgttgccccatggagaatgggcttcaggaagaatctgcctcagtcattttccagaagtgcctgagaggtgcagggcccgagtctggttaggtgacagcgggtcaagaggaggacattgtcctctctcttgtgttgctgcagatgccacaaaactgggccacggcacgatcaggcagttctggggcccccaggagggcagccttggggtgggcacagccagccctggcaggatgagcaactctgggtgggggggagtgggggatgagcagggggacatgaagatgcttggtggagcctggggtcatggctggtatctggttcctcccctgtgattccttcttggggactccaagacaggacaaggaagactggagccctccagaaacagatgggcccaggtccgtaagctgaggattcagtcccccctggattcaagcccagcatgtgcctacccagccagatgctccatgaacacagttcagggggcccgaagacagcactgttacctggagcccacatacagcctcagcaggcaaagggagactcagagaaaacatgggagggatttacagtcacatgcaggcagggaccagctcaacccttctttaatgtcatccagggagggggccagggatggaggggaggggttgaggagcgagaggcagttatttttgggtgggattcaccacttttcccatgaagaggggagacttggtattttgttcaatcattaagaagacaaagggtttgttgaacttgacctcgggggggatagacatgggtatggcctctaaaaacatggccccagcagcttcagtccctttctcgtcgatggtcagcacagccttatgcacggccttggagagcttcaggggtgcctcctctgtgaccccggagaggtcagccccattgctgaagaccttagtgatgcccagttgacccaggacgctcttcagatcataggttccagtaatggacagtttgggtaaatgtaagctggcagaccttctgtcttcattttccaggaacttggtgatgatatcgtgggtgagttcattttccaggtgctgtagtttcccctcatcaggcaggaagaagatggcggtggcattgcccaggtatttcatcagcagcacccagctggacagcttcttacagtgctggatgttaaacatgcctaaacgcttcatcataggcaccttcacggtggtcacctggtccacgtggaagtcctcttcctcggtgtccttgacttcaaagggtctctcccatttgcctttaaagaagatgtaattcaccagagcaaaaactgtgtctctgtcaagctccttgaccaaatccacaattttcccttgagtacccttctccacgtaatcgttgatctgtttcttggcctcttcggtgtccccgaagttgacagtgaaggcttctgagtggtacaactttttaacatcctccaaaaacttatccactagcttcaggccctcgctgaggaacaggccattgccggtggtcagctggagctggctgtctggctggttgagggtacggaggagttcctggaagccttcatggatctgagcctccggaatctccgtgaggttgaaattcaggccctccaggatttcatcgtgagtgtcagccttggtccccagggagagcattgcaaaggctgtagcgatgctcactggggagaagaagatattggtgctgttggactggtgtgccagctggcggtataggctgaaggcgaactcagccaggttgggggtgatcttgttgaaggttgggtgatcctgatcatggtgggatgtatctgtcttctgggcagcatctccctggggatcctcagccagggagacagggaccaggcagcacaggcctgccagcaggaggatgccccacgagacagaagacggcattgtcgattcactgtcccaggtcagtggtggtgcctgaagctgaggagacagggccctgtcctcgtccgtatttaagcagtggatccagaggggcaacgggggaggcgaaaggagtcattgtacctggctcagaaaccacagcgtcctgtgtccaaggtggagggggtggcgtgagtcagacagtctctgggagagtaccacttagctggccctctgctctcactgcagaatccttagtggctgttccactggtagcaagatctaccatttactgagtcaccccaaaatgcctgatgctgaagacttactgccgccctttcctgctctcctcaagctctcctcaagctctgtctcttctggcaggcacaggagagtggcctgaaggctggcaggaggttgccgcccctccaacctggaattcctggcagcagcagcggctaggccttcctcggaggcccgaccccctcctccttcttggttcagctcaggactctgagggttgctgcgtggaggcagtgcatgccctgggcacagtgcccagttcctgcccaSEQ ID NO: 23: Reverse complement of SEQ ID NO: 10tgcaagaaatgtagttctatttattctctgttctaatgggtataaacattttgttatctaacttgaacatcataccaactcaaaggcaggtcttactagcaatgactggggctcagagaggtttggcgacttcacgaaggtcacacagctgtcagggggaaaagtcagaacttggatccaggtcttcagactctcaggtctggtgtcatcctagggggcttggtgatggccatatctttaatgtatttgtggagagtgaaaggctgtcagtgagtaagcttaagagaacaggagacttgtgtgggaaacagtcggtatccattgattagactgaatcatgtagaattgctaatttcaccattttgaactatcgaaatcactatttggtatgactcaacctcatcctttaagtacacattcatgacagtgagtggttagacagtgattcctagattagtttgggatggggcagtgccttccataggacaaggccattcctggtagagacggagggagcaggctgtccttcagctaggggcccaggggacttcctggctgctgtgtcctttagcagggccttggaggatggaaggactctcctggcccttaccacaggggctattcaggaacagcctcctgccgtggcactggagctgcggtggccctctgaagactgcagggacagcaacaggcacaaagaagtcaggctgcatgtggccccagtcgggactcagaggaggaaagggaggggttgcgggggtcaatgggtgatgtgcttcctctcccatagctgaggagtccttgcaatggccttcctgagccatcagcaggcctatggccatgtgactagggaggagaagggatatagggtaatggtcttctggggcctgctgggcctggtcaagtctgaggaggagatagagaaaacaaagcagagaccctcctcttcatctggggagaagggacctgattctaaacggagatatgtgaggctttctggggcagcgatggaaggacaaggacagaataggtgtccttgttgccccatggagaatgggcttcaggaagaatctgcctcagtcattttccagaagtgcctgagaggtgcagggcccgagtctggttaggtgacagcgggtcaagaggaggacattgtcctctctcttgtgttgctgcagatgccacaaaactgggccacggcacgatcaggcagttctggggcccccaggagggcagccttggggtgggcacagccagccctggcaggatgagcaactctgggtgggggggagtgggggatgagcagggggacatgaagatgcttggtggagcctggggtcatggctggtatctggttcctcccctgtgattccttcttggggactccaagacaggacaaggaagactggagccctccagaaacagatgggcccaggtccgtaagctgaggattcagtcccccctggattcaagcccagcatgtgcctacccagccagatgctccatgaacacagttcagggggcccgaagacagcactgttacctggagcccacatacagcctcagcaggcaaagggagactcagagaaaacatgggagggatttacagtcacatgcaggcagggaccagctcaacccttctttaatgtcatccagggagggggccagggatggaggggaggggttgaggagcgagaggcagttatttttgggtgggattcaccacttttcccatgaagaggggagacttggtattttgttcaatcattaagaagacaaagggtttgttgaacttgacctcgggggggatagacatgggtatggcctctaaaaacatggccccagcagcttcagtccctttctcgtcgatggtcagcacagccttatgcacggccttggagagcttcaggggtgcctcctctgtgaccccggagaggtcagccccattgctgaagaccttagtgatgcccagttgacccaggacgctcttcagatcataggttccagtaatggacagtttgggtaaatgtaagctggcagaccttctgtcttcattttccaggaacttggtgatgatatcgtgggtgagttcattttccaggtgctgtagtttcccctcatcaggcaggaagaagatggcggtggcattgcccaggtatttcatcagcagcacccagctggacagcttcttacagtgctggatgttaaacatgcctaaacgcttcatcataggcaccttcacggtggtcacctggtccacgtggaagtcctcttcctcggtgtccttgacttcaaagggtctctcccatttgcctttaaagaagatgtaattcaccagagcaaaaactgtgtctctgtcaagctccttgaccaaatccacaattttcccttgagtacccttctccacgtaatcgttgatctgtttcttggcctcttcggtgtccccgaagttgacagtgaaggcttctgagtggtacaactttttaacatcctccaaaaacttatccactagcttcaggccctcgctgaggaacaggccattgccggtggtcagctggagctggctgtctggctggttgagggtacggaggagttcctggaagccttcatggatctgagcctccggaatctccgtgaggttgaaattcaggccctccaggatttcatcgtgagtgtcagccttggtccccagggagagcattgcaaaggctgtagcgatgctcactggggagaagaagatattggtgctgttggactggtgtgccagctggcggtataggctgaaggcgaactcagccaggttgggggtgatcttgttgaaggttgggtgatcctgatcatggtgggatgtatctgtcttctgggcagcatctccctggggatcctcagccagggagacagggaccaggcagcacaggcctgccagcaggaggatgccccacgagacagaagacggcattgtcgattcactgtcccaggtcagtggtggtgcctgaagctgaggagacagggccctgtcctcgtccgtatttaagcagtggatccagaggggcaacgggggaggctgctttcctgctctcctcaagctctcctcaagctctgtctcttctggcaggcacaggagagtggcctgaaggctggcaggaggttgccgcccctccaacctggaattcctggcagcagcagcggctaggccttcctcggaggcccgaccccctcctccttcttggttcagctcaggactctgagggttgctgcgtggaggcagtgcatgccctgggcacagtgcccagttcctgccca SEQ ID NO: 24: Reverse complement of SEQ ID NO: 11tgcaagaaatgtagttctatttattctctgttctaatgggtataaacattttgttatctaacttgaacatcataccaactcaaaggcaggtcttactagcaatgactggggctcagagaggtttggcgacttcacgaaggtcacacagctgtcagggggaaaagtcagaacttggatccaggtcttcagactctcaggtctggtgtcatcctagggggcttggtgatggccatatctttaatgtatttgtggagagtgaaaggctgtcagtgagtaagcttaagagaacaggagacttgtgtgggaaacagtcggtatccattgattagactgaatcatgtagaattgctaatttcaccattttgaactatcgaaatcactatttggtatgactcaacctcatcctttaagtacacattcatgacagtgagtggttagacagtgattcctagattagtttgggatggggcagtgccttccataggacaaggccattcctggtagagacggagggagcaggctgtccttcagctaggggcccaggggacttcctggctgctgtgtcctttagcagggccttggaggatggaaggactctcctggcccttaccacaggggctattcaggaacagcctcctgccgtggcactggagctgcggtggccctctgaagactgcagggacagcaacaggcacaaagaagtcaggctgcatgtggccccagtcgggactcagaggaggaaagggaggggttgcgggggtcaatgggtgatgtgcttcctctcccatagctgaggagtccttgcaatggccttcctgagccatcagcaggcctatggccatgtgactagggaggagaagggatatagggtaatggtcttctggggcctgctgggcctggtcaagtctgaggaggagatagagaaaacaaagcagagaccctcctcttcatctggggagaagggacctgattctaaacggagatatgtgaggctttctggggcagcgatggaaggacaaggacagaataggtgtccttgttgccccatggagaatgggcttcaggaagaatctgcctcagtcattttccagaagtgcctgagaggtgcagggcccgagtctggttaggtgacagcgggtcaagaggaggacattgtcctctctcttgtgttgctgcagatgccacaaaactgggccacggcacgatcaggcagttctggggcccccaggagggcagccttggggtgggcacagccagccctggcaggatgagcaactctgggtgggggggagtgggggatgagcagggggacatgaagatgcttggtggagcctggggtcatggctggtatctggttcctcccctgtgattccttcttggggactccaagacaggacaaggaagactggagccctccagaaacagatgggcccaggtccgtaagctgaggattcagtcccccctggattcaagcccagcatgtgcctacccagccagatgctccatgaacacagttcagggggcccgaagacagcactgttacctggagcccacatacagcctcagcaggcaaagggagactcagagaaaacatgggagggatttacagtcacatgcaggcagggaccagctcaacccttctttaatgtcatccagggagggggccagggatggaggggaggggttgaggagcgagaggcagttatttttgggtgggattcaccacttttcccatgaagaggggagacttggtattttgttcaatcattaagaagacaaagggtttgttgaacttgacctcgggggggatagacatgggtatggcctctaaaaacatggccccagcagcttcagtccctttctcgtcgatggtcagcacagccttatgcacggccttggagagcttcaggggtgcctcctctgtgaccccggagaggtcagccccattgctgaagaccttagtgatgcccagttgacccaggacgctcttcagatcataggttccagtaatggacagtttgggtaaatgtaagctggcagaccttctgtcttcattttccaggaacttggtgatgatatcgtgggtgagttcattttccaggtgctgtagtttcccctcatcaggcaggaagaagatggcggtggcattgcccaggtatttcatcagcagcacccagctggacagcttcttacagtgctggatgttaaacatgcctaaacgcttcatcataggcaccttcacggtggtcacctggtccacgtggaagtcctcttcctcggtgtccttgacttcaaagggtctctcccatttgcctttaaagaagatgtaattcaccagagcaaaaactgtgtctctgtcaagctccttgaccaaatccacaattttcccttgagtacccttctccacgtaatcgttgatctgtttcttggcctcttcggtgtccccgaagttgacagtgaaggcttctgagtggtacaactttttaacatcctccaaaaacttatccactagcttcaggccctcgctgaggaacaggccattgccggtggtcagctggagctggctgtctggctggttgagggtacggaggagttcctggaagccttcatggatctgagcctccggaatctccgtgaggttgaaattcaggccctccaggatttcatcgtgagtgtcagccttggtccccagggagagcattgcaaaggctgtagcgatgctcactggggagaagaagatattggtgctgttggactggtgtgccagctggcggtataggctgaaggcgaactcagccaggttgggggtgatcttgttgaaggttgggtgatcctgatcatggtgggatgtatctgtcttctgggcagcatctccctggggatcctcagccagggagacagggaccaggcagcacaggcctgccagcaggaggatgccccacgagacagaagacggcattgtcgattcactgtcccaggtcagtggtggtgcctgaagctgaggagacagggccctgtcctcgtccgtatttaagcagtggatccagaggggcaacgggggaggctttcctgctctcctcaagctctcctcaagctctgtctcttctggcaggcacaggagagtggcctgaaggctggcaggaggttgccgcccctccaacctggaattcctggcagcagcagcggctaggccttcctcggaggcccgaccccctcctccttcttggttcagctcaggactctgagggttgctgcgtggaggcagtgcatgccctgggcacagtgcccagttcctgcccaSEQ ID NO: 25 AAVALLPAVLLALLAP SEQ ID NO: 26 AALLPVLLAAP HIV Tat peptideSEQ ID NO: 27 GRKKRRQRRRPPQ Drosophila Antennapedia peptideSEQ ID NO: 28 RQIKIWFQNRRNIKWKK

What is claimed herein is:
 1. A double-stranded ribonucleic acid (dsRNA)for inhibiting expression of Serpina1, wherein said dsRNA comprises asense strand and an antisense strand, the antisense strand comprising aregion of complementarity which comprises at least 15 contiguousnucleotides differing by no more than 3 nucleotides from one of theantisense sequences listed in Tables 3 and
 4. 2. The dsRNA of claim 1,wherein the sense and antisense strands comprise sequences selected fromthe group composed of AD-44715.1, AD-44722.1, AD-44734.1, AD-44717.1,AD-44723.1, AD-44735.1, AD-44724.1, AD-44719.1, and AD-44737.1 of Table3.
 3. The dsRNA of claim 1, wherein said dsRNA comprises at least onemodified nucleotide.
 4. The dsRNA of claim 1, wherein at least one ofsaid modified nucleotides is chosen from the group of: a 2′-O-methylmodified nucleotide, a nucleotide comprising a 5′-phosphorothioategroup, and a terminal nucleotide linked to a cholesteryl derivative ordodecanoic acid bisdecylamide group.
 5. The dsRNA of claim 1, whereinsaid modified nucleotide is chosen from the group of: a2′-deoxy-2′-fluoro modified nucleotide, a 2′-deoxy-modified nucleotide,a locked nucleotide, an abasic nucleotide, 2′-amino-modified nucleotide,2′-alkyl-modified nucleotide, morpholino nucleotide, a phosphoramidate,and a non-natural base comprising nucleotide.
 6. The dsRNA of claim 1,wherein the region of complementarity is at least 17 nucleotides inlength.
 7. The dsRNA of claim 1, wherein the region of complementarityis between 19 and 21 nucleotides in length.
 8. The dsRNA of claim 7,wherein the region of complementarity is 19 nucleotides in length. 9.The dsRNA of claim 1, wherein each strand is no more than 30 nucleotidesin length.
 10. The dsRNA of claim 1, wherein at least one strandcomprises a 3′ overhang of at least 1 nucleotide.
 11. The dsRNA of claim1, further comprising a ligand.
 12. The dsRNA of claim 11, wherein theligand is conjugated to the 3′ end of the sense strand of the dsRNA. 13.The dsRNA of claim 1, wherein the region of complementarity consists ofone of the antisense sequences of Tables 3 and
 4. 14. The dsRNA of claim1, wherein the dsRNA comprises a sense strand consisting of a sensestrand sequence selected from Tables 3 and 4, and an antisense strandconsisting of an antisense sequence selected from Tables 3 and
 4. 15. Acell containing the dsRNA of claim
 1. 16. A vector encoding at least onestrand of a dsRNA of claim
 1. 17. A pharmaceutical composition forinhibiting expression of a Serpina1 gene comprising the dsRNA ofclaim
 1. 18. The pharmaceutical composition of claim 17, furthercomprising a lipid formulation.
 19. The pharmaceutical composition ofclaim 18, wherein the lipid formulation is a SNALP, or XTC formulation.20. A method of inhibiting Serpina1 expression in a cell, the methodcomprising: (a) introducing into the cell a double-stranded ribonucleicacid (dsRNA) comprising a sense strand and an antisense strand, theantisense strand comprising a region of complementarity which comprisesat least 15 contiguous nucleotides differing by no more than 3nucleotides from one of the antisense sequences listed in Tables 3 and4; and (b) maintaining the cell produced in step (a) for a timesufficient to obtain degradation of the mRNA transcript of a Serpina1gene, thereby inhibiting expression of the Serpina1 gene in the cell.21. The method of claim 20, wherein the Serpina1 expression is inhibitedby at least 30%.
 22. A method of treating a disorder mediated bySerpina1 expression comprising administering to a patient in need ofsuch treatment a therapeutically effective amount of a double-strandedribonucleic acid (dsRNA) comprising a sense strand and an antisensestrand, the antisense strand comprising a region of complementaritywhich comprises at least 15 contiguous nucleotides differing by no morethan 3 nucleotides from one of the antisense sequences listed in Tables3 and
 4. 23. The method of claim 22, wherein the disorder is Alpha 1anti-trypsin deficiency liver disease.
 24. The method of claim 22,wherein the administration of the dsRNA to the subject causes a decreasein cirrohsis, fibrosis, and/or Serpina1 protein accumulation in theliver.
 25. The method of claim 22, wherein the likelihood ofhepatocellular carcinoma occurring in the patient is reduced.
 26. Themethod of claim 22, wherein the dsRNA is administered at a concentrationof 0.01 mg/kg-5 mg/kg bodyweight of the patient.